Perl's Prussian Research Articles

Magnetic labeling of non‐phagocytic adherent cells with iron oxide nanoparticles: a comprehensive study

Small particles of iron oxide (SPIO) and ultrasmall particles of iron oxide (USPIO), inducing a strong negative contrast on T(2) and T(2)*-weighted MR images, are the most commonly used systems for the magnetic labeling of cultured cells and their subsequent detection by magnetic resonance imaging (MRI). The purpose of this work is to study the influence of iron incubation concentration, nanoparticle size and nanoparticle coating on the magnetic labeling and the viability of non-phagocytic adherent cells in culture. The magnetic labeling of 3T6 fibroblasts was studied by T(2)-weighted MRI at 4.7 T and by dosing-or cytochemical revealing-of iron through methods based on Perl's Prussian blue staining. Cells were incubated for 48 h with increasing iron concentrations of SPIO (25-1000 microg Fe/ml Endorem. Sinerem, a USPIO (20-40 nm) coated with neutral dextran, and Resovist (65 nm), a SPIO bearing an anionic carboxydextran coating, were compared with Endorem (dextran-coated, 80-150 nm) as magnetic tags. The iron loading of marrow stromal cell primary cultures (MSCs) isolated from rat femurs was compared with that of 3T6 fibroblasts. The SPIO-labeling of cells with Endorem was found to be dependent on the iron incubation concentration. MSCs, more sparsely distributed in the culture, exhibited higher iron contents than more densely populated 3T6 fibroblast cultures. A larger iron loading was achieved with Resovist than with Endorem, which in turn was more efficient than Sinerem as a magnetic tag. The magnetic labeling of cultured non-phagocytic adherent cells with iron oxide nanoparticles was thus found to be dependent on the relative concentration of the magnetic tag and of the cells in culture, on the nanoparticle size, and on the coating type. The viability of cells, estimated by methods assessing cell membrane permeability, was not affected by magnetic labeling in the conditions used in this work.

Macrophage Activity in Infected Areas of an Experimental Vertebral Osteomyelitis Model: USPIO-enhanced MR Imaging—Feasibility Study

To prospectively evaluate ultrasmall superparamagnetic iron oxide (USPIO) magnetic resonance (MR) imaging for the depiction of macrophages in infected areas of an experimental rabbit vertebral osteomyelitis model. Lumbar vertebral osteomyelitis was induced in 10 rabbits with intradiscal injection of bacteria in a vertebral disk (test level) versus saline injection in another disk (control level). After a mean interval of 12 days, rabbits were imaged prior to and 24 hours after administration of USPIO. The MR imaging protocol included T1-weighted spin-echo, T2-weighted fast spin-echo, and T2*-weighted gradient-echo sequences. MR findings were compared with histologic findings (macrophage immunostaining and Perls Prussian blue staining). A Wilcoxon signed rank test was used to compare signal-to-noise ratio (SNR) results before and after USPIO administration. T1-weighted MR images of infected vertebral test levels obtained 24 hours after USPIO administration showed a significant increase in SNR (P = .005), whereas T2- and T2*-weighted images showed no significant changes in SNR (P = .14 and P = .87, respectively). Histologic examination results of infected areas demonstrated complete replacement of hematopoietic bone marrow by macrophage infiltration. Perls Prussian blue staining showed that some macrophages were iron loaded. T1- (P = .02), T2- (P = .04), and T2*-weighted (P = .04) images of control vertebrae showed a significant decrease in SNR. Histologic examination results confirmed the persistence of normal hematopoietic bone marrow without macrophage infiltration, which was reflected by more intensive Perls Prussian blue staining compared with that in infected areas. MR imaging can depict USPIO-loaded macrophage infiltration present in infected areas in an experimental rabbit model of vertebral osteomyelitis.

Iron-fortified parboiled rice – A novel solution to high iron density in rice-based diets

The present study pioneered an investigation of a novel and cost-effective approach to fortify Fe in rice and to greatly improve Fe nutrition in rice-based diets through parboiling, though it remains at its preliminary phase. Rice grains of seven cultivars were parboiled in deionised water containing different levels of Fe chelate made by mixing different proportions of Fe sulfate (FeSO4) with ethylenediaminetetra-acetic acid disodium salt (Na2EDTA). Adding Fe to the parboiling water resulted in an increased Fe concentration in the most grain, effectively where FeSO4 and Na2EDTA were mixed at 2:1 molar ratio (11.16g Fe per 100g raw paddy grain). This treatment resulted in Fe concentrations in white rice milled for 60s and 120s, which were 20–50 times higher than those in the unfortified milled raw rice grains. The Fe concentrations in milled rice grains were 50–150mg Fe kg−1 in 60s milled grains with a slight reduction in 120s milled grains. Perls Prussian blue staining of the cross section of Fe-fortified parboiled rice grains suggested inward movement of added Fe into the endosperm through the apoplastic pathway in the dorsal region of the rice grain. The retention rates of fortified Fe varied among the different cultivars, possibly due to different physical–chemical properties of the grains. The percentages of soluble fraction of the total Fe were higher than 50% in all cultivars tested, indicating its high bioavailability potential, though it remains to be evaluated. The present findings provided a preliminary basis for further investigation of this innovative technique, before its adoption by parboiled rice industry, such as optimising the levels of Fe addition and industrial process and Fe bioavailability in Fe-fortified-parboiled rice.

Hfe Acts in Hepatocytes to Prevent Hemochromatosis

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.

The differentiation of anaemia in rheumatoid arthritis: parameters of iron-deficiency in an Indian rheumatoid arthritis population

Iron deficiency anaemia (IDA) is common in Indian patients with rheumatoid arthritis (RA). We evaluated red blood cell indices, serum iron related and bone marrow iron stores measurements in diagnosing iron deficiency in patients with RA. Fifty consecutive anaemic patients with RA had their complete blood counts, red cell indices, serum iron, serum ferritin and serum total iron binding capacity (TIBC) measured and underwent posterior iliac crest bone marrow aspiration. Fixed smears were stained for iron with Perl's Prussian blue and patients who had no (grade 0) or minimal stainable iron (grade I) were regarded as iron deficient and rest iron replete (grade II-IV) and hence as having anaemia of chronic disease (ACD). To determine diagnostic power of tests used for diagnosing iron deficiency in addition to positive likelihood ratio, sensitivity, specificity and negative predictive values; receiver operating characteristics (ROC) curves were plotted and areas under the receiver-operating curves were compared. Eighteen patients (36%) had IDA and 32 (64%) had ACD. Correlation between the bone marrow iron stores and serum ferritin was poor in the IDA group (r = -0.09, P = 0.57) and significant in the ACD group (r = 0.79, P < 0.0001). Areas under the ROC curves for mean corpuscular haemoglobin (MCV), serum iron, TIBC and mean corpuscular haemoglobin concentration (MCHC) were relatively low (0.52, 0.71, 0.75 and 0.77, respectively) and these tests had low positive likelihood ratios (1.08, 2.13, 4.62 and 1.5, respectively). Both area under ROC curve [0.98, 95% confidence interval (0.94, 0.99)] and negative predictive value (97%) were highest when cut off serum ferritin was <82 microg/l. In patients with RA serum iron, TIBC, MCV and MCHC have limited value in diagnosing iron deficiency. In this study compared to American and European studies a much higher cut off value of serum ferritin had most diagnostic power for detecting iron deficiency. Bone marrow iron stores measurements appears to be the most reliable method for diagnosing IDA however, it needs to be taken in conjunction with other laboratory findings and the clinical scenario.

Iron‐oxide labeling of hematogenous macrophages in a model of experimental autoimmune encephalomyelitis and the contribution to signal loss in fast imaging employing steady state acquisition (FIESTA) images

To determine the contribution of blood-derived macrophages to the signal loss observed in MR images of inflammatory lesions in experimental autoimmune encephalomyelitis (EAE). A relapsing-remitting form of EAE was induced in transgenic mice that express enhanced green fluorescent protein (EGFP) specifically in hematopoietic cells of the myelomonocytic lineage. Animals were injected with Feridex, a superparamagnetic iron oxide (SPIO) nanoparticle, 24 hours prior to in vivo MRI. MRI was performed using a 1.5T whole-body scanner; a high-performance, custom-built gradient coil insert; and a 3D steady-state free precession (SSFP) imaging pulse sequence. Comparisons were made between MR images and corresponding anti-GFP and Perl's Prussian blue (PPB)-stained brain sections. MR images revealed the presence of discrete regions of signal loss throughout the brains of EAE animals that were administered Feridex. Histological staining showed that regions of signal loss on MR images corresponded anatomically with regions of PPB- and GFP-positive cells. This experiment provides the first direct evidence that macrophages of hematogenous origin are labeled with SPIO after intravenous administration of Feridex, and contribute to the regions of signal loss detected in MR images of EAE brain.

Semi-quantitative analysis for selecting Fe- and Zn-dense genotypes of staple food crops

Four semi-quantitative screening methods were developed for plant breeding purposes to identify iron (Fe) and zinc (Zn)-dense genotypes in germplasm, elite lines and early generation progeny. Methods include colour image analysis for Fe and Zn in wheat and rice grains, and spectrophotometric analysis of Fe and Zn in ground flour of rice, wheat, potato, sweet potato and cassava. Staining with 71 mM Perl's Prussian blue solution (PPB) and subsequent image analysis with Adobe Photoshop ® to determine pixel numbers in the stained regions lead to the quantification of Fe. Due to differences in grain size between the genotypes evaluated, correlations between inductively coupled plasma-optical emission spectrophotometry (ICP-OES) Fe and PPB derived Fe were improved by standardizing according to grain weight. The ratio of total blue pixel number (TPN)/total grain weight (TGW) of 21 rice lines correlated ( r=0.84, p<0.001) with the Fe concentration derived by ICP-OES. Similarly, a colorimetric method was developed for Zn analysis using 1.56 mM Dithizone (DTZ) solution and subsequent quantification by image analysis with Adobe Photoshop ®. As with the Fe analysis, the ratio of TPN/TGW of 70 wheat lines correlated better with ICP-OES Zn analysis ( r=0.82, p<0.001) and successfully separated low and high Zn grain germplasm. Ground polished rice and wheat flour were spectrophotometrically analysed after simple extraction in 0.5 M HCl solution using a modified 2,2′-dipyridyl method for Fe, and a modified Zincon ® method for Zn. These two methods show good correlations with ICP analyses ( r=0.93 and 0.92 for Fe and Zn, respectively) and thus can be used for semi-quantitative screening to discriminate between genotypes that are either high or low in Fe or Zn. The more precise ICP-OES and AAS methods could then be used to quantify actual amounts of Fe and Zn in those genotypes identified as Fe- and Zn-dense from the initial screening.

Association of hepatic iron overload with invasive fungal infection in liver transplant recipients

Invasive fungal infection is a serious complication of orthotopic liver transplantation, but its risk factors remain incompletely defined. Iron overload has already been associated with increased risk of fungal infections, but it has not yet been assessed as a risk factor in liver transplantation. We retrospectively studied a cohort of 153 consecutive patients who underwent their first liver transplantation at a single center and who survived at least 7 days after transplantation. The association between various pretransplant patient characteristics, including hepatic explant iron and risk of invasive fungal infections, was analyzed by univariate and multivariate models. Iron in the hepatic explant was assessed by Perl's Prussian blue stain by a pathologist blinded to clinical outcome. During the first year after transplantation, 28 of 153 patients developed a total of 31 invasive fungal infections, of which 21 (68%) were caused by Candida, 7 (23%) by Aspergillus, 2 (6%) by Cryptococcus, and 1 (3%) by Saccharomyces. Stainable iron in the hepatic explant was found in 48 patients (31%). Stainable iron in the hepatic explant was found to be strongly and independently associated with posttransplantation fungal infections in multivariate analysis (hazard ratio 3.09; 95% confidence interval 1.45-6.56; P = 0.003). Hepatic iron overload is strongly and independently associated with posttransplantation invasive fungal infections. Studies to confirm this finding in other centers and define the mechanism are warranted.

Magnetic Targeting of Magnetoliposomes to Solid Tumors with MR Imaging Monitoring in Mice: Feasibility

To establish the feasibility of magnetoliposome tumor targeting with an extracorporeal magnet. Animal experiments were performed in compliance with Institut National de la Santé Et de la Recherche Médicale animal protection guidelines and were approved by local government authorities. Magnetophoresis was used to measure the velocity of magnetoliposomes constituted of polyethylene glycol-lipids and anionic maghemite nanocrystals in a calibrated magnetic field in vitro. For in vivo studies, 38 male Swiss nude mice bearing a PC3 human prostate carcinoma tumor in each flank received an intravenous injection of magnetoliposomes (n = 27), saline (n = 9), or nonencapsulated superparamagnetic particles (n = 2) after a small magnet with a magnetic field of 0.3 T and a field gradient of 11 T/m was fixed to the skin above one tumor. The animals were examined at magnetic resonance (MR) imaging with eight different sequences, iron doses (13 mice), and magnet-application durations (12 mice). Their excised tumors were then stained with Perls Prussian blue and hematoxylin-eosin and were examined histologically. With use of the paired Student t test, signal intensity, tumor surface enhancement, and number of stained cells were compared between the control and magnet-exposed tumors to determine significant differences (P </= .01). The mean magnetoliposome velocity ranged from 10 to 40 mum/sec when the magnetic field equaled 0.13 T and the field gradient equaled 25 T/m. At T1-weighted three-dimensional spoiled gradient-echo MR imaging in vivo, the tumor exposed to the magnet showed strong negative enhancement, -52%, compared with the -7% enhancement of the other tumor. Maximal enhancement occurred after 3 hours of magnet application. After 24 hours of magnet application, intracapillary iron particle accumulation was observed in the targeted tumors only. Magnetic targeting of sterically stabilized magnetoliposomes after they are intravenously injected is feasible in vivo.

Macroscopic and Microscopic Aspects of Collared Peccary and White-lipped Peccary Placenta

This study examines middle and late gestational placentae from 13 Tayassu tajacu (collared peccary) and 3 Tayassu pecari (white-lipped peccary), which are Artiodactyla belonging to the Family Tayassuidae. The chorionic sac of Tayassu species is diffuse and chorioallantoic. These epitheliochorial placentae show no trophoblast invasion into the uterine epithelium and there is interdigitation between fetal and maternal microvilli. Two distinct regions of the fetomaternal interface can be identified: the interareolar and the areolar regions. The uterine epithelium has eosinophilic cytoplasm with dispersed, basophilic and electron-dense granules. Trophoblast cells are irregularly cuboidal on top of the fetal ridges and columnar on troughs, where cells have cytoplasmic vesicles and large basal vacuoles, surrounded by whorls of smooth membranes. Capillaries indent the trophoblast cells forming a placental barrier 3 microm or less thick. The columnar uterine glandular epithelium has a subpopulation of granules staining with Perl's Prussian blue reaction, suggesting iron secretion. In areolar areas, the trophoblast cells show apical microvilli, a basophilic cytoplasm with electron-dense intracellular vacuoles and cisternae. The placenta can therefore be classified as non-deciduate. The ultrastructural aspects of this study reveal features that have not previously been described and extend our knowledge of functions relating to materno-fetal transport in these species.

Up-regulation of transferrin receptor 1 in chronic hepatitis C: Implication in excess hepatic iron accumulation

: To clarify the mechanism of excess hepatic iron accumulation in chronic hepatitis C, we investigated the expressions of transferrin receptor 1 and divalent metal transporter 1 in hepatocytes, both of which are involved in cellular iron uptake, in relation to the degree of hepatic iron accumulation and hepatic fibrosis by immunohistochemistrical study. : Forty-six hepatic tissues with chronic hepatitis C and five normal hepatic tissues were examined. Chemical detection of hepatic iron accumulation was performed by Perl's Prussian blue stain. The immunohistochemistrical study was performed by avidin-biotin complex method with alkaline phosphatase. : In chronic hepatitis C: (1) Hepatic iron accumulation was significantly increased in relation to the advance of the fibrosis. (2) Divalent metal transporter 1 decreased significantly in relation to the advance of hepatic fibrosis. (3) Transferrin receptor 1 expression was always detected, although not in normal hepatic tissues; there was no relation between expression levels and the degree of hepatic fibrosis. : These data demonstrated that the transferrin receptor 1 expression was up-regulated irrespective of the degree of hepatic iron accumulation, suggesting that the up-regulation of transferrin receptor 1 might act as one of the key mechanisms implicated in the accumulation of hepatic iron in chronic hepatitis C.

Complexities in the Rat Iron Loading Model for Pre-Clinical Testing of Iron Chelators.

Transfusion-dependent iron overload, such as occurs in beta-thalassaemia (Cooley's Anaemia), leads to lethal cardiac toxicity in the second decade of life if not treated by iron chelation, but even with subcutaneous desferrioxamine (DFO) cardiac disease remains a problem, although delayed by 1–2 decades. As we design novel iron chelators, we are testing them in various animal models of iron overload. While assessing outcomes we have observed relatively sparse reports of systematic studies on organs, tissues, cellular, or subcellular iron distribution. Therefore we initiated a series of studies to characterize iron distribution using various approaches. Multiple intraperitoneal injections of iron dextran, 200 mg/kg/week X 4 − 16 weeks, followed by an equilibration period of minimum 1–2 weeks was studied as a means of increasing total body iron load in hundreds of rats under various conditions. Sacrifice varied from 6 weeks to 1 year post iron loading and the concentration of iron in liver, heart, and other tissues, organs, cells and subcellular organelles was examined. Quantitatively, in untreated rats (no chelators), the liver/heart iron ratio was about 10:1, consistent with the accumulation observed in post-mortem studies in humans prior to extensive use of iron chelation. Much less-well described has been the distribution of iron in lymphatic tissues. Our studies revealed that lymph nodes become visibly enlarged. In addition, randomly distributed brown spots appeared in the omentum. Such changes persisted up to one year after iron loading, regardless whether they were treated daily with chelators (DFO or deferiprone) in standard doses for four months. Even after a single intraperitoneal iron-dextran injection of 200 mg/kg, changes were visible. Histopathological analysis (hematoxilin-eosin for general histology and Perl's Prussian Blue for iron) showed extensive iron accumulation in the omentum, and in the cortical and subcortical regions of the enlarged lymph nodes. Electron microscopy revealed cellular (macrophages) and subcellular (mitochondria) iron localization in the lymph nodes. When iron was administered as iron sucrose (single ip dose), iron accumulation was more extensive in the omentum and in the peritoneal fat in comparison to iron dextran, but the enlargement of the lymph nodes was not observed. Quantitative iron measurement (via validated HPLC method) in the liver and heart after a single iron dextran (N=30, up to 29th day) and iron sucrose dose (N=6 up to 50th day) was in agreement with the histological observations. Iron accumulation in the omentum and lymph nodes after four months of chelation treatment and one year after iron loading indicated the resistance of these unusual iron “pools” to chelation therapy. These studies confirm that different iron formulations may result in different patterns of iron distribution and they also raise questions about the suitability of rats as an animal model for transfusional iron overload in humans.

Dietary aluminium and renal failure in the koala (Phascolarctos cinereus).

The study investigated the link between the potentially nephrotoxic levels of aluminium ingested in the natural diet of eucalypt leaves by koalas in the Adelaide Hills, South Australia, and the high incidence of renal failure in koalas within this habitat. Routine histology of kidney specimens revealed no pathologies at the light microscopic level and contrasted sharply with the clinical signs of renal failure. However staining with solochrome azurine and Perl's Prussian blue showed aluminium was present in some proximal convoluted tubules in all specimens. Aluminium was also found in bone samples. The presence of aluminium in bone and kidney tissues was confirmed using electron dispersive x-ray analysis with transmission and scanning electron microscopy. Ultrastructural changes, including a decrease in lysosomal numbers, were seen in proximal convoluted tubules and these changes were shown to coincide with the presence of aluminium. No aluminium was found in koalas that died from causes other than renal failure. It was concluded that renal failure in the koalas of the Adelaide Hills is characterised by the presence of aluminium in the kidneys and bone and it is probably related to the high levels of aluminium in their restricted diet of eucalypt leaves. However, it is not known if the presence of aluminium is the cause or effect of the renal failure. The study is the first account where aluminium ingested as part of the natural diet of mammals has been shown to accumulate in the animal and be implicated with nephrotoxicity.

DMT1 genetic variability is not responsible for phenotype variability in hereditary hemochromatosis

Background/aims. Homozygosity for a cysteine to tyrosine translocation at position 282 within the HFE gene (C282Y) is responsible for over 90% of hereditary hemochromatosis (HH) in Celtic populations. Determining those C282Y homozygotes at greatest risk for iron overload is a major clinical concern as only a small percentage will develop clinically significant iron overload. Divalent metal transport protein (DMT1) on the apical surface of duodenal enterocytes is recognised as the major iron import protein. We investigated whether genetic variability within the DMT1 gene may partly explain the phenotypic variability seen amongst a group of C282Y homozygotes with iron overload. Methods. One hundred and one unrelated C282Y homozygotes and 103 C282Y negative controls were analysed for the presence of four specific mutations/polymorphisms within the DMT1 gene ( 1245T/C, 1303C/A, IVS4 + 44C/A, IVS15Ex16-16C/G) using standard PCR techniques. Hepatic iron deposition was determined in 32 HH patients following Perls Prussian blue staining (0–4+). Estimations of the haplotype frequencies were performed utilising the program Arlequin version 2. Results. There were no significant differences in the allele frequencies of the IVS4 + 44C/A, 1303C/A, 1254T/C and IVS15Ex16-16C/G polymorphisms in the patient cohort compared to those observed in the control cohort. The commonest haplotypes identified were CCTC: IVS4C + 44C, 1303C, 1254T, IVS15ex16-16C; ACCC: IVS4C + 44A, 1303C, 1254C, IVS15ex16-16C and ACTG: IVS4C + 44A, 1303C, 1254T, IVS15ex16-16G. Similarly, there were no significant differences in the frequencies of these three haplotypes in the patient cohorts (regardless of the degree of hepatic iron deposition) compared to the control cohort. Conclusions. Polymorphisms within DMT1 gene do not influence penetrance of the HH phenotype.

Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane.

To investigate whether iron is involved in the pathogenesis of age-related macular degeneration (AMD). Postmortem AMD-affected (nonexudative or exudative) and healthy maculas were studied using the 3,3'-diaminobenzidine-enhanced Perls Prussian blue stain. The Perls Prussian blue stain was quantified by computer-assisted analysis of digital images. To determine whether the iron was chelatable, sections treated with the iron chelator deferoxamine were compared with adjacent, nonchelated sections. Compared with healthy maculas, AMD-affected maculas had statistically significant increases in the total iron level. Some of this iron was chelatable. The iron was present in retinal pigment epithelium and Bruch's membrane in maculas from patients who had drusen only, geographic atrophy, and exudative AMD in pathologic areas and, occasionally, in relatively healthy areas. Oxidative stress has been implicated in the pathogenesis of AMD by the Age-Related Eye Disease Study. Increased concentrations of iron, which generate highly reactive hydroxyl radicals via the Fenton reaction, may induce oxidative stress in the macula and lead to AMD. As the increased iron concentrations in AMD-affected eyes consist in part of a chelatable iron pool, treatment of patients who have AMD with iron chelators might be considered a potential therapy. While there are, as yet, no clinical data indicating that the treatment of patients who have AMD with iron chelators is beneficial, data presented herein indicate that further investigation of iron concentrations in postmortem tissues and the mechanisms of iron transport in the retina is warranted.

Image Processing to Facilitate Histological Evaluation of Tissue Specimens Stained With Perl's Prussian Blue

Image-processing techniques were developed to assist histological analysis of rat spleen specimens stained for iron with Perl's Prussian blue. The stained areas in the splenic tissue images were segmented on the basis of color attributes. A series of image features was extracted to describe the subjective concept of blueness, an important attribute in the histological evaluation of blue-stained tissues. Feature extraction was based on statistical analysis. A neural network model was developed to predict pathologists' scores from selected image features. The model predicted pathologists' scores with an R 2 value of 0.86.

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 cytocen‐trifuge 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 &lt; .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.

Sideroblastic anaemias.

Sideroblastic anaemias.

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.

Abundance of Siderophages in Sputum

To investigate the lung response to traffic-related air pollution by enumerating hemosiderin-laden alveolar macrophages (AM) in sputum. Sputum samples were collected from 103 urban adult males from Calcutta chronically exposed to automobile exhaust. Forty-nine rural individuals served as controls. AM were identified by nonspecific esterase staining. Perl's Prussian blue technique was employed for the detection of hemosiderin-laden AM (siderophages). The urban group, consisting of 31 traffic officers, 25 automobile service station workers and 47 street hawkers, had seven times more AM in their sputum than did the matched controls. Besides, a remarkable rise (27-fold) in the number of siderophages in sputum was observed in urban individuals. Smoking further elevated the AM count and number of siderophages. Abundant siderophages in the urban group may indicate the toxic effect of airborne pollutants on the lung, leading to phagocytosis of destroyed cells, including erythrocytes, and accumulation of iron in AM. Enumeration of siderophages in sputum appears to be a simple, noninvasive, inexpensive cytochemical technique well suited to preliminary assessment of the adverse effects of air pollution on the lungs in large, population-based studies, especially in developing countries.