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.     

A Versatile New Mineralized Bone Stain for Simultaneous Assessment of Tetracycline and Osteoid Seams

A versatile mineralized bone stain (MIBS) for demonstrating osteoid seams and tetracycline fluorescence simultaneously in thin or thick undecalcified sections has been developed. Bone specimens are fixed in 70% ethanol, but 10% buffered formalin is permissible. Depending upon one's preference, these specimens can be left unstained or be prestained before plastic embedding. Osteoid seams are stained green to jade green, or light to dark purple. Mineralized bone matrix is unstained or green. Osteoblast and osteoclast nuclei are light to dark purple, cytoplasm varies from slightly gray to pink. The identification of osteoid seams by this method agrees closely with identification by in vivo tetracycline uptake using the same section from the same biopsy. The method demonstrates halo volumes, an abnormal, lacunar, low density bone around viable osteocytes in purple. This phenomenon is commonly seen in vitamin D-resistant rickets, fluorosis, renal osteodystrophy, hyperparathyroidism, and is sometimes seen in fluoride treated osteoporotic patients. In osteomalacic bone, most osteoid seams are irregularly stained as indicated by the presence of unmineralized osteoid between mineralized lamellae. The method has been used effectively in staining new bone formation in hydroxyapatite implants and bone grafts. Old, unstained, plastic embedded undecalcified sections are stained as well as fresh sections after removal of the coverslip. This stain also promises to be valuable in the study of different metabolic bone diseases from the point of view of remodeling, histomorphometry, and pathology.     

Time-lapsed microstructural imaging of bone failure behavior

Many bones within the axial and appendicular skeleton are subjected to repetitive loading during the course of ordinary daily activities. If this loading is of sufficient magnitude or duration, failure of the bone tissue may result. Until recently the structural analysis of these fractures has been limited to two-dimensional sections. Due to the inherent destructiveness of this method, dynamic assessment of fracture progression has not been possible. An image-guided technique to analyze structural failure has been developed utilizing step-wise micro-compression in combination with time-lapsed micro-computed tomographic imaging. This technique allows, for the first time, direct three-dimensional visualization and quantification of fracture initiation and progression on the microscopic level and relates the global failure properties of trabecular bone to those of the individual trabeculae. The goals of this project were first to design and fabricate a novel micro-mechanical testing system, composed of a micro-compression device and a material testing and data acquisition system; and second, to validate the testing system to perform step-wise testing of trabecular bone specimens based on image-guided failure analysis. Due to the rate dependant properties of bone, stress relaxation was a concerning factor with respect to the step-wise testing method. In order to address these concerns, the results of the step-wise testing method were compared to those obtained from a conventional continuous test (considered to be the gold standard for the step-wise compressive mechanical testing) over the same total strain range and testing conditions. This was performed using porous aluminum alloy samples with highly reproducible and homogenous structural properties as well as trabecular bone samples from a single whale vertebra. Five cylinders from aluminum foam and trabecular whale bone each were compressed and imaged in a step-wise fashion from 0% to 20% strain at intervals of 2%, 4%, 8%, 12%, 16% and 20%. Mechanical properties obtained from the continuous and step-wise methods were not significantly different for both aluminum foam and whale bone specimens (p>0.05). Both testing methods yielded very similar stress-strain graphs with almost identical elastic and plastic regions with overlaying standard error bars for both whale bone and aluminum foam specimens. This was further concurred by performing regression analyses between the stress data from both testing methods (r(2)=0.98 for whale bone and aluminum foam specimens). Animations of fracture initiation and progression revealed that failure always occurred in local bands with the remaining regions of the structure largely unaffected independent of structure type. In conclusion, we found step-wise micro-compression to be a valid approach for image-guided failure assessment (IGFA) with high precision and accuracy as compared to classical continuous testing. We expect findings from upcoming studies of IGFA of human vertebral bone to improve our understanding of the relative importance of densitometric, morphological, and loading factors in the etiology of spontaneous fractures of the spine. Eventually, this improved understanding may lead to more successful approaches to the prevention of age-related fatigue fractures.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity,validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics.

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

Large Specimen Bone Embedment and Cement Line Staining

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.     

Softening Hair with Dithiothreitol (Cleland's Reagent) for Histological Sectioning in Paraffin

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.     

Changes in mechanical properties of bone within the mandibular condyle with age

The purpose of the study was to compare indentation modulus (IM) and hardness of condylar bone in young and adult dogs. In addition we desired to examine histologic sections for bone formation activity in the two groups. Mandibular condyles were obtained from adult (1- to 2-year-old) and young (approximately 5-m old) dogs. Two sections/condyle were obtained and one was processed for histomorphometry and the other for mechanical analyses. Indents were made on moist condylar trabecular bone to a depth of 500 nm at a loading rate of 10 nm/s using a custom-made hydration system to obtain IM and hardness. Histomorphometric analyses measured the bone volume/total volume (BV/TV%) and ratio of labeled to unlabeled bone within the condyle. Data were analyzed using a repeated-measures factorial analysis of variance and Tukey-Kramer method. Overall, the IM of the adult condyles (10.0+/-3.4 GPa, Mean+/-SD) were significantly (P<0.0001) higher than in young dogs (5.6+/-2.6 GPa). There was a greater bone mass in the young (60.2%) versus the adult condyles (42%). Also, significantly more labeled bone in the young (66.1%) condylar bone suggested higher bone forming activity than in adult condyles (27.5%). With age there is a change in mass and material properties in the trabecular bone of the mandibular condyle in dogs.     

Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting.

Recently two highly sensitive and specific diagnostic criteria for Sj?gren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure.     

A Three-Dimensional Histological Method for Direct Determination of the Number of Trabecular Termini in Cancellous Bone

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 fjim thick slices of methylmethacrylate embedded material rather than the more usual thin 8 μm. histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, “real” (i. e., unstained) trabecular termini can be distinguished from “apparent” (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphom-etry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.     

Silver Staining of Bone Prior to Decalcification for Quantitative Determination of Osteoid in Sections

Slices, 1-2 mm thick, of alcohol-fixed bone are immersed in 2% aqueous AgNO₃ in the dark for 48 hr. After thorough washing in running tap water, the silver phosphate formed at the interface of osteoid and calcified bone is reduced to a black deposit by 5% aqueous sodium hypophosphite containing 0.1 N NaOH, 0.2 ml/100 ml. The blocks are then immersed in 5% aqueous Na₂S₂O₃ and after further washing pass through a routine formic acid decalcification and paraffin wax embedding schedule. Sections cut at 5 μ thickness and counterstained with Van Gieson's picrofuchsin show a clear differentiation between osteoid tissue and the outer limit of calcification in trabecular or cortical bone, thus making them suitable for quantitative studies. The main advantage of the method is the production of intact stained sections without specialised embedding or cutting techniques.     

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.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.     

Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.     

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.     

Laser Microprobe Mass Analysis (LAMMA) to Verify the Aluminon Staining of Bone

Triammonium aurin tricarboxylate (aluminon) has been used to localize aluminum in 2 μm 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.