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Electron microscopy of hemosiderin; presence of ferritin and occurrence of crystalline lattices in hemosiderin deposits.

Injections of hemoglobin were given to rats in order to produce hemosiderosis, and selected hemosiderin granules in sectioned cells of proximal convoluted tubules were studied by means of electron microscopy. When examined at high resolution, many of the dense particles that were present in hemosiderin granules proved to have the structure that characterizes the iron hydroxide micelles of molecular ferritin. In some hemosiderin deposits the dense particles formed lattices similar to those present in sections of crystalline ferritin. Such ordered arrangement of dense particles was encountered inside as well as outside of the cytoplasmic organelles for which the name "siderosomes" has been proposed previously, and which may be derived from mitochondria. Study of hemosiderin granules in hepatic parenchymal and reticuloendothelial cells of human beings yielded similar results. The findings confirm the inference that ferritin is a component of hemosiderin, and they indicate that some of the so called hemosiderin granules are crystals of ferritin.

Animals↗

Quantitative assessment of a change of hemosiderin deposition with age in splenic compartments of rats.

Hemosiderin deposition was quantitatively estimated in paraffin sections stained using Perls' iron reaction in the spleens of untreated male rats aged from 0 to 28 months. Hemosiderin was deposited in the periarteriolar lymphocyte sheath (PALS), lymph follicles, and marginal zone, as well as in the red pulp which was the main compartment of hemosiderin deposition. Special attention was paid to the PALS and marginal zone. Marked hemosiderin deposition was evident from 4 months, although only few hemosiderin-laden macrophages appeared in the first 3 months. Hemosiderin deposition peaked at 12 months in the red pulp and at 20 months in the white pulp and marginal zone. The deposition in the white pulp and marginal zone was about 1/9 to 1/4 of that in the red pulp. A few hemosiderin-laden macrophages were found in the lymph follicles after 2 months. In the periarterial region of the splenic hilus, deposition of hemosiderin granules was observed even at 1 month when there was little hemosiderin deposition elsewhere. Since no arteries open there, the presence of hemosiderin-laden macrophages in the PALS and lymph follicles suggested movement of some macrophages from the red pulp and/or the marginal zone. In addition, hemosiderin deposition in the PALS increased with age after it decreased in the other compartments.

Aging↗

Relation between bone marrow hemosiderin iron, serum iron status markers, and chemical and histochemical liver iron content in 82 patients with alcoholic and nonalcoholic hepatic disease.

Bone marrow hemosiderin iron was assessed in 48 patients with alcoholic, and in 34 patients with nonalcoholic liver disease (53 men, 29 women, median age 55 years, range 18-84) and correlated to serum (S)-iron status markers (iron, transferrin, ferritin), as well as to histochemical hepatocyte iron and chemical liver iron content. In a control group of 53 healthy subjects (23 men, 30 women, median age 28 years, range 18-90) marrow hemosiderin iron and iron status markers were evaluated as well. Among liver patients, the marrow iron grade was higher in men than in women (p = 0.03). Correlations were found between marrow iron and histochemical liver iron (rho = 0.38, p = 0.0001) as well as chemical liver iron (rho = 0.33, p = 0.01). Marrow iron was correlated to S-ferritin (rho = 0.53, p = 0.0001), mean red cell volume (rho = 0.34, p = 0.003), and S-transferrin (rho = -0.24, p = 0.02). Alcoholics had a higher marrow iron grade than nonalcoholics (p = 0.001) and controls (p = 0.0001). Among controls, the marrow iron grade was likewise higher in men than in women (p = 0.01). Correlations were found between marrow iron and ferritin (rho = 0.64, p = 0.0001), transferrin saturation (rho = 0.56, p = 0.001), transferrin (rho = 0.53, p = 0.001), S-iron (rho = 0.37, p = 0.01), and hemoglobin in women (rho = 0.38, p = 0.05). The results indicate that alcoholics either have increased marrow hemosiderin iron stores, or display a redistribution of iron in reticuloendothelial cells from soluble ferritin-bound iron to insoluble hemosiderin iron. Among patients with absent marrow hemosiderin iron, 81% had absent hepatocyte hemosiderin iron as well. Among patients with absent hepatocyte hemosiderin iron, 23% had absent and 77% normal or increased marrow hemosiderin iron. Therefore, in patients with iron depletion, assessment of marrow hemosiderin iron yields more relevant information of iron status than assessment of hepatocyte hemosiderin iron.

Adolescent↗

Urine hemosiderin: a novel marker to assess the severity of chronic venous disease.

OBJECTIVE: Impaired venous drainage in severe chronic venous insufficiency (CVI) leads to microcirculatory overload, characterized by erythrocyte diapedesis and subsequent extravascular hemolysis, resulting in typical dermal hemosiderin deposition. We hypothesized that hemosiderin, normally absent, could be present in the urine in CVI. METHODS: The three-phase study included 117 patients with CVI and 12 healthy control subjects, all of whom had undergone clinical examination and duplex scanning. In phase 1, current methods were used to test urine for hemosiderin in 61 persons: 12 healthy control subjects, 24 patients with mild CVI (clinical class C1 to C3), and 25 patients with severe CVI (clinical class C4 to C6). In phase 2, the concentration of urinary hemosiderin was determined in 45 consecutive patients with CVI, CEAP class 1 to 6. A score of 0 was assigned when typical hemosiderin granules were absent at microscopic examination, a score of 1 when one to three granules per field were detected; 2 when four to six granules were detected; and 3 when more than six granules were observed. Phase 3 included 23 patients with CVI (clinical class 2 to 6). Hemosiderin concentration was determined and a score assigned before patients underwent surgical procedures to correct primary CVI. Both hemosiderin testing and duplex scanning were repeated after 6 months. RESULTS: Phase 1: Urine hemosiderin testing to determine presence or absence of CVI in patients with reflux detectable at duplex scanning yielded the following values: positive predictive value, 96% (95% confidence interval [CI], 86% to 100%); negative predictive value, 88% (CI, 68% to 97%); sensitivity, 94% (CI, 72% to 99%); specificity, 91% (CI, 83% to 99%); and diagnostic accuracy, 95% (CI, 86% to 99%). Phase 2: Hemosiderinuria score enabled classification of clinical severity of CVI. Mean scores, respectively, were clinical class 1, 0.18 +/- 0.12; class 2, 0.75 +/- 0.47; class 3, 1.67 +/- 0.21; class 4, 1.86 +/- 0.26; class 5, 2.50 +/- 0.28; and class 6, 1.92 +/- 0.21 (P <.001). Phase 3: At 6-month follow-up, hemosiderin score was improved, from 2.48 +/- 0.12 preoperatively to 0.78 +/- 0.18 postoperatively (P <.0001). A score of 0 or 1 was associated with successful surgery, whereas a score of 2 or 3 reflected persistence of reflux. CONCLUSIONS: Determination of presence of hemosiderin in the urine is a new, sensitive, cost-effective, noninvasive, and repeatable test that enables detection of substantial microcirculatory overload in patients with CVI.

Chronic Disease↗

The nature of storage iron in idiopathic hemochromatosis and in hemosiderosis. Electron optical, chemical, and serologic studies on isolated hemosiderin granules.

Using three different methods of cells fractionation, hemosiderin granules were isolated from tissues (liver and/or spleen) of three patients. The samples were obtained from a case of idiopathic hemochromatosis, a case of thalassemia major with secondary (transfusional?) hemosiderosis, and a case of transfusional hemosiderosis associated with an unclassified anemia. Iron, nitrogen, and protein content of the hemosiderin granules varied over a wide range. Electron microscopy of sectioned granules revealed aggregates of dense particles of different shapes, with diameters ranging from 10 A to about 75 A. In some of the granules dense particles corresponding to the iron hydroxide micelles of ferritin molecules were abundant. But many of the granules contained very few of these molecules. The presence of ferritin and apoferritin in the samples of hemosiderin granules was demonstrated by means of precipitin tests in agar-gel, using rabbit antiferritin sera with known antibody nitrogen concentrations. At least three antigenic components were detected in highly purified crystalline ferritin prepared from tissues of the three patients; the hemosiderin granules contained the same antigens, but probably in much smaller quantities. Both ferritin and apoferritin molecules were extracted from hemosiderin granules, and were demonstrated in the electron microscope after suitable preparation. The solubility curve of human ferritin in solutions of (NH(4))(2)SO(4) was investigated. The results indicate that substantial quantities of ferritin or apoferritin can be lost in saline, aqueous media during isolation of hemosiderin granules from cells. It was shown by means of electron microdiffraction on selected hemosiderin granules that the dense particles represent forms of partly hydrated alpha-Fe(2)O(3). The conditions necessary for electron microdiffraction in an electron microscope precluded an exact determination of the state of hydration of the alpha-Fe(2)O(3) or of its structural relation to (FeOOH) micelles of pure ferritin in its undenatured state. The findings were considered in the light of evidence on the structure and disposition of hemosiderin in situ in cells, and on the structure of ferritin. Differences between endogenous hemosiderin and hemosiderin derived from injections of colloidal iron compounds were pointed out. The evidence indicates that in hemochromatosis and in secondary hemosiderosis much of the inorganic storage iron in liver and spleen is derived from degraded ferritin. The findings suggest that an abnormal cellular metabolic pathway of ferritin is implicated in the pathogenesis of hemochromatosis and transfusional hemosiderosis.

Anemia↗

A study of hemosiderosis with the aid of electron microscopy; with observations on the relationship between hemosiderin and ferritin.

Hemosiderin deposits in rats and in man were studied and compared by means of electron and light microscopy. Typical, isotropic, iron-positive hemosiderin granules were found to contain innumerable, closely packed, electron-dense particles, embedded in matter that was much less dense to electrons. Similar dense particles were often scattered diffusely through the cytoplasmic matrix of cells containing hemosiderin granules. In cells of proximal convoluted tubules of rats given repeated intraperitoneal injections of hemoglobin the hemosiderin granules contained dense particles with a mean diameter of 55 A, and with a size-frequency distribution that indicated uniformity. These particles corresponded in size to the iron micelles of ferritin molecules. There was less uniformity of particles in hemosiderin granules situated in liver and reticulo-endothelial cells of rats that had been given a diet containing ethionine. The dense aggregates representing hemosiderin granules were often situated inside discrete cytoplasmic organelles that were bordered by membranes, and sometimes contained "cristae"; and often the membranous borders were markedly disrupted. The term "sidersomes" is proposed for these specialized cytoplasmic structures which may be derivatives of mitochondria, and apparently play a part in the formation of hemosiderin. Ferritin was crystallized from the livers and kidneys of the hemosiderotic rats with ease, but could not be crystallized from comparable quantities of liver and kidney tissue of untreated control rats. Specimens from the liver and spleen of a patient with advanced hemosiderosis, obtained at an operation, were also studied. In liver and reticulo-endothelial cells many particles with diameters of about 60 A were scattered through the cytoplasmic matrix. By contrast, hemosiderin granules in the same cells contained particles that varied considerably in size. In representative granules, examined at high resolution, the size-frequency distribution of particle diameters displayed a periodicity consistent with the presence of small, uniform subunits. Electron micrographs of ferritin, isolated from the spleen of the same patient, provided confirmation for the inferences that the dense particles observed inside cells are iron micelles, and that ferritin is probably a component of hemosiderin.

Animals↗

Detection of hemosiderin deposition by T2*-weighted MRI after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is very difficult to diagnose several months after its onset. We thus investigated subarachnoid hemosiderin deposition well after SAH by T2*-weighted MRI, a sensitive method for hemosiderin detection. METHODS: To investigate how hemosiderin deposition as confirmed by T2*-weighted MRI contributes to the determination of prior SAH and how the extent of hemosiderin deposition is associated with a number of clinical factors, we retrospectively analyzed 58 patients >3 months after SAH associated with ruptured aneurysms. We also investigated 209 healthy volunteers as controls. RESULTS: T2*-weighted MRI demonstrated subarachnoid hemosiderin deposition in 72.4% of the SAH patients, whereas no deposition was seen in the healthy volunteer group. The hemosiderin was preferentially deposited in the subarachnoid space near a ruptured aneurysm. Odds ratios (ORs) were estimated from logistic regression analyses correlating hemosiderin deposition with other factors. Age (>or=54 years) (OR, 5.1; 95% CI, 1.03 to 25.0; P=0.046), Fisher grade 3 on initial CT (OR, 8.0; 95% CI, 1.26 to 50.4; P=0.027), and Karnofsky Scale score <or=80% 6 months after onset of SAH (OR, 12.8; 95% CI, 1.97 to 83.3; P=0.0077) were all found to be independently associated with hemosiderin deposition levels. CONCLUSIONS: T2*-weighted MRI is an effective means of diagnosing prior SAH and may also reveal the location of a ruptured aneurysm. The extent of hemosiderin deposition was significantly associated with several factors, including age, CT findings, and poor prognosis.

Adult↗

Assessment of pulmonary and intrathymic hemosiderin deposition in sudden infant death syndrome.

The aim of this study was to stain lung and thymus gland sections that had been taken from infants who had died of sudden infant death syndrome (SIDS) for interstitial hemosiderin and to compare the results with those obtained for controls. There were two groups of SIDS infants, one with, and a second group without, histories of apparent life-threatening events (ALTEs). No significant difference in numbers of cases with interstitial hemosiderin deposition was found between SIDS infants with histories of ALTEs (n = 4 of 12, 33.3%), SIDS infants without histories of ALTEs (n = 4 of 22, 18.2%), and controls (n = 4 of 24, 16.7%). However, if four of the control cases with histories of previous chest trauma were excluded, there was a significantly greater number of cases with pulmonary interstitial hemosiderin in the SIDS infants with histories of ALTEs compared with the subgroup of control infants with no previous chest trauma (n = 1 of 20, 5%) (P < .05). No relationship could be established between the timing of the ALTEs, the type of resuscitation or age of the infant at death, and the presence of hemosiderin. None of the sections of thymus gland stained positively for hemosiderin. Positive staining for pulmonary interstitial hemosiderin, therefore, differentiated a group of SIDS infants with histories of previous ALTEs from a subgroup of control infants with no histories of previous chest trauma. However, pulmonary interstitial hemosiderin staining could not be used with certainly to confirm or exclude previous ALTEs in individual SIDS cases as not every SIDS case with a history of an ALTE stained for pulmonary interstitial hemosiderin. In addition, positive staining occurred for SIDS infants without histories of ALTEs and also for control infants who died of other causes.

Case-Control Studies↗

Iron absorption by humans from hemosiderin and ferritin, further studies.

Iron absorption from hemosiderin and ferritin biosynthetically labeled with radioactive iron has been studied in 61 subjects. The geometrical mean iron absorption from hemosiderin in both normal and iron deficient subjects was 3.4%. Its mean absorption ranged from 1.9% in normal subjects to 4.7% in subjects with moderate iron deficiency and 7.3% in subjects with marked iron deficiency. The iron absorption from hemosiderin was markedly increased when it was administered with ascorbic acid or liver. The absorption of iron from hemosiderin when hemosiderin and wheat were consumed in a meal, was lower than the absorption from wheat. Iron from liver ferritin and liver hemosiderin were less absorbed in this study than that previously reported for liver hemoglobin. The studies presented here support the possibility that ferritin and hemosiderin form an iron pool different from the non-heme pool formed by vegetal iron, egg iron and ferric and ferrous salts.

Absorption↗

Image analysis of hemosiderin-laden macrophages in bronchoalveolar lavage fluid.

The aim of this study was to assess quantitatively the relationship between the number of iron-laden macrophages, extent of hemosiderin content and amount of dark-stained hemosiderin clumps in bronchoalveolar lavage (BAL) fluids. The series included specimens from 20 subjects whose BAL had yielded iron-laden macrophages. Six had developed hemoptysis 3-15 days prior to BAL. Quantitative studies were performed on smears stained by the ferrocyanide reaction, using true color image analysis for image segmentation. A top-hat function based on mathematical morphologic concepts was used for extraction of dark clumps of hemosiderin. The results demonstrated the presence of iron-laden macrophages three days after hemoptysis. There was a moderate correlation between the percentage of macrophages and amount of hemosiderin content, but the extent of dark-stained clumps did not increase in parallel to the increase in hemosiderin content and probably represented an advanced stage in the phagocytic processing of hemosiderin formed from engulfed red blood cells. The main difference between subjects with and without previous hemoptysis was higher values for dark clumps of hemosiderin in the former.

Adult↗

Morphometrical analysis of hemosiderin deposits in relation to wound age.

A morphometrical analysis of the extent of hemosiderin deposits in 71 human skin wounds with post-infliction intervals between 2 days and 7 months was performed. Earliest positive findings were detectable in a lesion aged 3 days, and with increasing wound age an increase in the amount of hemosiderin occurred. A value of more than 20% of the microscopic field with hemosiderin deposits was found earliest 8 days after wounding and therefore the detection of considerable amounts of hemosiderin (arbitrarily defined as 20% or more of the evaluated area) indicates a minimum wound age of approximately 1 week. Since the extent of hemosiderin formation depends upon the extent of the initial hemorrhage and a "physiological" reduction in the amount of this pigment with advanced wound age, slight or absent hemosiderin deposits cannot provide information on the post-infliction interval.

Adult↗

Seizure outcome after resection of cavernous malformations is better when surrounding hemosiderin-stained brain also is removed.

PURPOSE: Considering the epileptogenic effect of cavernoma-surrounding hemosiderin, assumptions are made that resection only of the cavernoma itself may not be sufficient as treatment of symptomatic epilepsy in patients with cavernous malformations. The purpose of this study was to test the hypothesis whether seizure outcome after removal of cavernous malformations may be related to the extent of resection of surrounding hemosiderin-stained brain tissue. METHODS: In this retrospective study, 31 consecutive patients with pharmacotherapy-refractory epilepsy due to a cavernous malformation were included. In all patients, cavernomas were resected, and all patients underwent pre- and postoperative magnetic resonance imaging (MRI). We grouped patients according to MRI findings (hemosiderin completely removed versus not/partially removed) and compared seizure outcome (as assessed by the Engel Outcome Classification score) between the two groups. RESULTS: Three years after resection of cavernomas, patients in whom hemosiderin-stained brain tissue had been removed completely had a better chance for a favorable long-term seizure outcome compared with those with detectable postoperative hemosiderin (p=0.037). CONCLUSIONS: Our study suggests that complete removal of cavernoma-surrounding hemosiderin-stained brain tissue may improve epileptic outcome after resection of cavernous malformations.

Adolescent↗

MR findings indicative of hemosiderin in giant-cell tumor of bone: frequency, cause, and diagnostic significance.

OBJECTIVE: We studied the frequency, cause, and diagnostic significance of MR findings indicative of large amounts of hemosiderin in giant-cell tumor of bone. MATERIALS AND METHODS: The clinical, MR, and histologic findings in 16 patients with giant-cell tumors of bone were reviewed. Eight tumors occurred in uncommon locations or in patients who were not in the usual age range; the other eight were more typical. Areas of decreased MR signal intensity considered to be caused by hemosiderin were identified. The number and distribution of RBCs within the tumor were studied histologically, and the presence of hemosiderin was determined on histologic sections made with iron stains. RESULTS: In 10 cases in which MR images showed areas of low signal intensity, large amounts of hemosiderin were noted histologically. The low-signal-intensity areas were nodular, zonal, whorled, or diffuse and occupied at least one fifth of the tumor volume. On histologic examination, many erythrocytes were in direct contact with the tumor cells, and the hemosiderin was seen in both mononuclear and multinuclear tumor cells. CONCLUSION: Hemosiderin is commonly seen on MR images of giant-cell tumors of bone (63%) and is probably related to the extravasated erythrocytes in the tumor and the phagocytic function of the tumor cells. This MR finding supports the diagnosis, even in giant-cell tumors in uncommon locations or in patients who are not in the typical age range.

Adult↗

Time course of hemosiderin production and clearance by human pulmonary macrophages.

Tracheal aspirates from four previously healthy infants with acute pulmonary hemorrhage, and small volume bronchial lavages from children undergoing flexible fiberoptic bronchoscopy were examined for pulmonary alveolar macrophages (PAM) containing hemosiderin. Hemosiderin formation was also studied in vitro. Macrophages containing hemosiderin were first seen in tracheal aspirates 50 hours after an acute pulmonary hemorrhage and after 72 hours in cultured macrophages. A small percentage of the PAM recovered by bronchoalveolar lavage from both adults and children contained hemosiderin. Hemosiderin was rapidly cleared from the lungs following an acute pulmonary hemorrhage.

Female↗

Alveolar macrophage graded hemosiderin score from bronchoalveolar lavage in horses with exercise-induced pulmonary hemorrhage and controls.

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise-induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2-5 years was used. Horses were grouped as either control (EIPH-negative) or EIPH-positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocentrifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P < .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa = 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.

Animals↗

Hemosiderin-laden macrophages in bronchoalveolar lavage fluid.

Diffuse pulmonary hemorrhage syndromes occasionally present diagnostic problems if the clinical picture is not very typical. The presence of hemosiderin-laden alveolar macrophages in bronchoalveolar lavage (BAL) fluid is a useful diagnostic criterion for this entity. However, in many diffuse interstitial pulmonary diseases (DIPD) there is a lesion at the alveolocapillary barrier, and an exit of red blood cells could occur from the blood vessels, leading to the appearance of siderophages. The aim of this work was dual: to evaluate the presence and number of siderophages in different types of interstitial pulmonary disease and to compare the diagnostic yield of two ways of quantifying hemosiderin-laden macrophages. Three groups of patients--controls (n = 5), DIPD (n = 32) and diffuse pulmonary hemorrhage (n = 3)--were subjected to BAL, and a differential count was made on cytocentrifuged Diff-Quik- and Perl-stained preparations. On the latter, two different measurements were made: the number of macrophages laden with hemosiderin and a quantitative "score." The results of a conventional count (percent of Perl-positive macrophages) showed significant differences between the three groups considered overall. Applying a cutoff value of 20%, the sensitivity of this method was 100% and the specificity, 91.6%. The results of the hemosiderin score showed significant differences between the three groups. Applying a value of 50 as a cutoff, the sensitivity and specificity of the method were 100%. In control patients and carriers of DIPD, the percentage of alveolar macrophages was higher than in healthy subjects. Quantification of the hemosiderin content of alveolar macrophages improved the specificity of the diagnosis of diffuse pulmonary hemorrhage by BAL.

Bronchoalveolar Lavage Fluid↗