Laser microprobe mass analysis (LAMMA) to verify the aluminon staining of bone

Triammonium aurin tricarboxylate (aluminon) has been used to localize aluminum in 2 micron sections of undecalcified, methyl methacrylate embedded bone obtained from patients with terminal chronic renal failure. Aluminum appeared in four cases as bright red lines at the mineralized-bone boundary. In two cases, however, purplish lines were found and one patient showed red as well as purplish lines. Laser microprobe mass analysis (LAMMA) identified aluminum at the location of the red lines and both aluminum and iron at the purplish lines. Furthermore, both iron and aluminum were found in histiocytic bone marrow cells, which showed brownish aluminon staining. It appears that when aluminum and iron occur together, aluminon staining may yield aberrant results. This study shows that LAMMA can be used for the identification of elements sought by histochemical methods and thus permits the evaluation of their staining effects.     

Use of laser microprobe mass analysis (LAMMA) for localizing multiple elements in soft and hard tissues

The potential of laser microprobe mass analysis (LAMMA) as a sensitive microanalytical technique was explored in applications relevant to nephrology. Aluminum and associated elements, such as iron, were localized in fresh tissue biopsies obtained from uremic patients treatment by chronic hemodialysis. The LAMMA was applied to serum, liver, bone, and parathyroid glands of such patients. In addition, we used LAMMA to evaluate the specificity and sensitivity of routine histochemistry, in particular on human bone sections stained by the aluminon method. The high, multielemental sensitivity and molecular microprobe potential of LAMMA established important advantages over other microchemical methods forin situ analysis at the micron level in histological sections.     

A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Aluminon: its limited application as a reagent for the detection of aluminum species

The triammonium salt of aurin tricarboxylic acid, commonly referred to as aluminon, forms a dye that has been used for the colorimetric determination of Al(III) species. We have reviewed the pertinent literature on the reaction of aluminon with respect to the metallic species that form colored aluminon complexes. The effects of experimental variables, such as time, temperature, and pH, upon the color development of the aluminon complex are also presented. Organic and inorganic species, particularly Be(II) and Fe(III), which can affect color formation, are described. The use of aluminon as a histochemical staining agent for the detection of aluminum requires verification by atomic absorption spectrophotometric analysis or other quantitative techniques.     

Histochemical demonstration of aluminum and iron deposition in pulmonary bony tissues in three cases of diffuse pulmonary ossification

Diffuse pulmonary ossification is a rare condition. We examined three cases of it in Japan, and attempted histochemically to stain for deposition of aluminum and iron in bony tissues. The patients were all female, and in their mid-twenties, mid- eighties, and later teen years. One of the patients had been exposed to heavy metals in her work involving heavy-metal analyses for 18 months. Aluminum staining and Berlin blue staining for iron were performed with dewaxed, undecalcified sections of pulmonary tissues from these three cases. Interestingly, all pulmonary bony tissues from the three cases examined exhibited linear regions of both aluminum and iron deposition in the calcifying fronts or the cement lines of bones. The patient exposed to heavy metals exhibited the most severe aluminum and iron deposition, and also exhibited positive reaction for both aluminum and iron in elastic fibers of blood vessels. Foreign body granulomas with multinucleated giant cells exhibiting elastophagia were also found in this case. This phenomenon, "endogenous pneumoconiosis", appeared to have been the cause of pulmonary hemorrhage in this case, resulting in focal heavy hemosiderosis. It is of great interest that identical patterns of aluminum and iron deposition in hemodialysis patients were found in these three cases, This is the first report on histochemical demonstration of aluminum and iron deposition in diffuse pulmonary ossification, and detailed analysis of additional cases is needed.     

The application of laser microprobe mass analysis to the study of biological material

Laser microprobe mass analysis (LAMMA) is an investigational method which is a powerful tool for the identification and quantitation of various elements present in small volumes of tissue. LAMMA is highly sensitive and capable of rapidly detecting concentrations of 1–3 p.p.m. of most metallic elements, in precisely localized cellular compartments. In order to further assess its value, cultured skin fibroblasts and biopsy tissues from human subjects and experimental animals were probed by LAMMA, and the results were correlated with ultrastructural findings. Biopsy samples were obtained from patients suffering from Gaucher disease, and from patients and animals with pathologic iron or copper metabolism. No significant abnormalities were detected in the cultured fibroblasts from patients with Gaucher disease, in contrast to the iron content of tissue biopsy Gaucher cells, which was markedly increased, apparently as a consequence of erythrophagocytosis. Particularly intense iron-related peaks were found in liver cytosiderosis due to neonatal or genetic haemochromatosis, thalassaemia major and in animal models of iron overload. An additional finding was the presence of aluminium accumulation in siderosomes of different cells. In liver biopsy samples from human Wilson's disease and from rats with an inherited disorder causing copper toxicosis, copper-containing compounds were identified and localized, and their relative concentration was estimated by LAMMA. The present study showed that LAMMA is a valuable technique for the localization and estimation of relative abundance of trace elements in various tissues containing excessive amounts of metals.     

Iron and aluminum lakes of Gallo blue E as nuclear and metachromatic mucin stains.

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Bone aluminum uptake in uremic rats receiving intraperitoneal iron

To assess the effect of concomitant iron and aluminum loads on bone aluminum accumulation and on the response to the deferoxamine test in rats with the same aluminum surcharge, Wistar rats with chronic renal failure were divided into three groups: iron-overloaded rats (N=6) (intraperitoneal iron); iron-depleted rats (N=6) (blood withdrawal two to three times per week); control rats (N=4) (no manipulation). All groups received intraperitoneal aluminum simultaneously. After 6 wk, a deferoxamine challenge test was performed. Thereafter, bone aluminum and iron were measured. The iron-overloaded rats showed higher bone iron content (iron overloaded: 147.7±55.4 μg/g; iron depleted: 7.9±1.0, and controls 13.3±9.9 μg/g, p<0.010) and lower bone aluminum content (iron overloaded: 14.2±4.0 μg/g; iron depleted: 70.9±35.1 μg/g; controls: 72.7±28.3 μg/g p<0.005). No differences were found between the iron-depleted and control rats. After the deferoxamine infusion, the iron-depleted rats tended to have higher serum aluminum increments (p=NS) and higher urinary aluminum excretion (p<0.012, p<0.020) than control rats despite similar amounts of aluminum in bone of the two groups. Aluminum bone accumulation was minor if iron and aluminum loads were given concomitantly. The iron depletion influenced the results of the deferoxamine challenge test in rats with similar bone aluminum burden.     

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

A combined stain for identifying epithelial cells of the gastric mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This adoring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast ells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent wed and the amount of Al²⁺ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.     

Characterization and Separation of Some of the Coordination Compounds of Chromium and Aluminon

Chromic nitrate and aluminon (ATA) react to form highly colored lakes. Examination of these lakes by the Vosburgh and Cooper method has led to the identification of a pigment [(ATA)Cr(H₂O)_x] which is insoluble in water and absolute alcohol, a deep red anionic component [(ATA)_BCr(H₂O)_x]^-y, and a purplish-red cationic component [(ATA)Cr₄(H₂O)_x]^+y. These components of the lake have been isolated and separated in the dry form. The anionic compound is apparently a simple coordination compound and can be used as a cytoplasmic stain while the cationic component is a chelate and can be used as a rather selective nuclear stain. In addition to these components, a number of other components were also found. Not only the ratio between chromium and aluminon but also the concentration of the reactants influences the formation of these different components. The amount of pigment formed is maximal with a molar ratio of 1 and a concentration of 10^-2 M or 10^-1 M. The anionic component is maximal with a molar ratio of 1 Cr to 6 aluminon at the 10^-1 M concentration, the cationic component is maximal with a molar ratio of 4 chromium to 1 aluminon at the 10^-1 M concentration. None of these are formed at the 10^-4 M concentration but only a deep redpurple soluble compound at the 1 : 1 ratio, which was not further investigated.     

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora)

Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.     

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora)

Summary Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

An Air Distributer for use in the Drop Method of Dehydration

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

A sensitive stain for aluminum in undecalcified cancellous bone

Aluminum is known to accumulate with age in bone and other tissues of humans, even in the absence of renal disease. Our study aimed to develop a histological staining method sufficiently sensitive to detect aluminum in plastic sections of undecalcified bone biopsies from healthy volunteers as well as from patients with renal and non-renal bone diseases. We used quantitative histomorphometry to measure the percentage of trabecular surface stained by aluminum and found that our new method was approximately 50% more sensitive for detecting aluminum than the Acid Solochrome Azurine (ASA) method which in turn was significantly more sensitive than the Aluminon method. Aluminon is widely used in pathology laboratories for diagnostic purposes despite concerns in the literature about Aluminon's limited sensitivity for aluminum. Our histomorphometric results showed that the newly developed method stained approximately 10% of the trabecular surface in bone sections from healthy controls, 38% from renal patients, 26% from patients with vitamin D deficiency, and 29% from patients with osteoporosis. Histomorphometric measurements of aluminum-stained trabecular surfaces in sections stained with ASA were consistent with those obtained in Walton-stained sections but proportionately lower. Moreover, the Walton and ASA methods stained aluminum at similar locations in adjacent bone sections. As the ASA and Walton methods are considerably more sensitive for bone aluminum than the Aluminon method, we recommend that either of them should be used in place of the Aluminon method for routine diagnostic purposes.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Decreased bone marrow iron in chronic granulocytic leukaemia: a consistent finding not reflecting iron deficiency

The iron status of 50 patients with Ph'-positive chronic granulocytic leukaemia (CGL) was evaluated at diagnosis by means of bone marrow and blood studies. A decreased or absent iron in semiquantitative estimation on bone marrow smears was observed in 92% of patients, and 88% had a low sideroblast score. In contrast, normal Hb and serum iron concentrations were found in the majority of cases, and only two out of the 50 patients displayed a decreased serum ferritin. To ascertain whether the bone marrow pattern of iron depletion could be due to an expansion of the red cell mass, the latter parameter was measured by isotopic methods in a subgroup of 11 patients. Normal or slightly increased values were obtained in all cases. We conclude that absent or decreased marrow iron is a common feature in the chronic phase of CGL, that generally does not reflect true iron deficiency. Since such a finding is also usual in polycythaemia vera and idiopathic myelofibrosis, it should be included among the features shared by the chronic myeloproliferative disorders.