Reproducibility of morphometric measurements of amyloid after various staining methods

Four histologic staining methods used for detecting amyloid (Congo red, viewed in both normal and polarized light, Sirius red, Crystal violet and Thioflavine T) were applied to heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The amount of amyloid present was evaluated with morphometry (point counting) by five pathologists, and the interobserver reproducibility and variation of point counting in these staining methods were analyzed. The Sirius red method showed the least variation and was the most suitable stain for demonstrating amyloid with respect to reproducibility. Thioflavine T showed the greatest variation and was the least suitable stain with respect to reproducibility. The range of variation was considerable in all staining methods. The results show that stains differ in their specificity and sensitivity in staining amyloid, observers differ in their interpretation of staining results and certain stains result in more uniform interpretations than do others.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)     

Characterization of spontaneous amyloidosis of Syrian hamsters using the potassium permanganate method

Amyloid from 16 Syrian hamsters with spontaneous age-related systemic amyloidosis retained affinity for Congo red dye after treatment with potassium permanganate. Serum electrophoresis showed a slight rise in gamma globulins without a monoclonal spike. Bence-Jones proteins were not present in the urine. There was no histological evidence of plasmacytosis. Amyloidosis could not be associated with chronic inflammation. The lack of apparent etiology of this amyloid and its resistance to potassium permanganate treatment suggests that it is a primary non-AA amyloid. This finding varies with a report suggesting that the spontaneous amyloidosis of aged Syrian hamsters is associated with the AA fibril.     

Trichrome Staining for Detection of Amyloid

It has been proposed to use trichrome staining of histological sections for the detection of connective tissue fiber and sites for amyloid localization, as well as for increasing color contrast. After incubation in acidin–pepsin solution, sections are dewaxed and successively stained with picrofuchsin according to van Gieson, together with nuclei counterstain with hematoxylin, Congo red, and picroindigocarmine. As a result, the amyloid bound with collagen fibers was stained brick-red, collagen and reticular fibers not bound with amyloid was stained blue-green, and cytoplasm of cells not containing amyloid was stained yellow. Trichrome staining of organs affected by amyloidosis is more informative for the analysis of organs than Congo red stain.     

Senile cardiac amyloid: demonstration of a unique fibril protein in tissue sections.

Antisera were raised against degrading amyloid fibrils isolated from the heart of a patient with senile cardiac amyloidosis (SCA), and from a medullary carcinoma of the thyroid (MCT). The antisera were absorbed and used in indirect immunofluorescence to identify an amyloid fibril protein (ASCA) in heart tissue from patients with senile cardiac amyloidosis and to identify the amyloid fibril protein (AMCT) found in association with medullary carcinomas of the thyroid. Absorbed anti-ASCA antiserum did not react with normal tissue such as heart, liver, spleen, and striated muscle, or with amyloid tissue known to contain amyloid fibril proteins AA, AlambdaI, AlambdaIV, AlambdaV, AMCT or with pancreatic tissue containing islet amyloid deposits. The reactions with senile amyloid he,rt tissue could be blocked completely by degraded amyloid fibrils extracted from senile amyloid heart tissue or by amyloid fibril protein ASCA isolated from such fibrils. The anti-AMCT antiserum showed a similar specific reaction restricted to amyloid associated with MCT. In addition, antisera specific for amyloid fibril proteins AA, AlambdaI, AlambdaIV, and AlambdaV failed to react with senile cardiac amyloid, pancreatic islet amyloid, or medullary thyroid amyloid.     

Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain

In the current protocol, we describe the Congo red staining method and a method for separately quantifying vascular and parenchymal amyloid deposits in brain tissue sections. Congo red staining detects amyloid deposits in brain tissue of amyloid precursor protein transgenic mice and human Alzheimer's tissue. It detects compacted amyloid in a beta-sheet secondary structure and labels amyloid in both the brain parenchyma (amyloid plaques) and blood vessels. Congophilic amyloid in blood vessels is called cerebral amyloid angiopathy (CAA). To date, analysis of CAA has largely used a severity rating scale, including both qualitative and quantitative characteristics. Here, we describe a simple method for quantifying total Congophilic staining and resolution of this staining into the parenchymal and vascular components based on morphological criteria. It is becoming increasingly important to separately quantify various components of the Alzheimer's pathology, given the advancement of amyloid-lowering therapies into clinical trials. The entire procedure for the Congo red staining can be performed at room temperature (20-25 degrees C) in a fume hood. The staining protocol should take 1 h 30 min including time for coverslipping slides. Time required for image analysis depends greatly on the number of samples being analyzed and the software being used. In our hands, 30 images can be collected per hour and quantified in a further 2 h.     

Amyloid in cytologic specimens. Differential diagnosis and diagnostic pitfalls.

OBJECTIVE: To describe and illustrate the characteristic features of amyloid in cytologic preparations and point out its diagnostic pitfalls. STUDY DESIGN: Five fine needle aspirates and one bronchial washing that contained amyloid were retrospectively reviewed. The aspirates were obtained from each of the five following sites: lung, occipital lymph node, thyroid gland, proximal humerus and subcutaneous soft tissue. Smears of all of the aspirates were stained with Papanicolaou stain, and in two cases they were also stained with Diff-Quik. Cell block sections were stained with hematoxylin and eosin. Congo red, CD45 and CD20 were used on selected cases. RESULTS: Amyloid appears as either flocculent material or irregularly shaped fragments with scalloped and pointed edges. The amorphous fragments are acellular and frequently associated with connective tissue cells. They stain eosinophilic to cyanophilic with Papanicolaou stain and deep blue with Diff-Quik. In two cases an exuberant giant cell reaction almost obscured the amyloid. In the thyroid aspirate, the amyloid was misinterpreted as colloid. In bronchial washings and lung aspirates, amyloid has to be distinguished from mucus, alveolar proteinosis, chondroid material and corpora amylacea. When circumferentially surrounded by lymphocytes or plasma cells, flocculent amyloid deposits may simulate adenoid cystic carcinoma. CONCLUSION: Amyloid can be easily overlooked or mistaken for other entities with similar staining qualities. Congo red staining can help to confirm the diagnosis.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

MALDI Mass Spectrometry Imaging: A Novel Tool for the Identification and Classification of Amyloidosis

Amyloidosis is a group of diseases caused by extracellular accumulation of fibrillar polypeptide aggregates. So far, diagnosis is performed by Congo red staining of tissue sections in combination with polarization microscopy. Subsequent identification of the causative protein by immunohistochemistry harbors some difficulties regarding sensitivity and specificity. Mass spectrometry based approaches have been demonstrated to constitute a reliable method to supplement typing of amyloidosis, but still depend on Congo red staining. In the present study, we used matrix‐assisted laser desorption/ionization mass spectrometry imaging coupled with ion mobility separation (MALDI‐IMS MSI) to investigate amyloid deposits in formalin‐fixed and paraffin‐embedded tissue samples. Utilizing a novel peptide filter method, we found a universal peptide signature for amyloidoses. Furthermore, differences in the peptide composition of ALλ and ATTR amyloid were revealed and used to build a reliable classification model. Integrating the peptide filter in MALDI‐IMS MSI analysis, we developed a bioinformatics workflow facilitating the identification and classification of amyloidosis in a less time and sample‐consuming experimental setup. Our findings demonstrate also the feasibility to investigate the amyloid's protein composition, thus paving the way to establish classification models for the diverse types of amyloidoses and to shed further light on the complex process of amyloidogenesis.     

Comparison of Sirius red and Congo red as stains for amyloid in animal tissues.

Sirius red and Congo red were compared for specificity and sensitivity of amyloid staining in animal and human material. Previously described advantages of Sirius red as an amyloid dye were confirmed, as well as its disadvantage of lack of ultraviolet fluorescence. Two further disadvantages of Sirius red were discovered, both relating to animal material: (a) its unexpectedly weak staining of early experimentally induced amyloid deposits and (b) frequent uncontrollable nonspecific staining of fibrous tissues. It is therefore concluded that, overall, Congo red used by the improved alkaline technique of Puchtler, Sweat and Levine (1962) remains the best single method for demonstration of amyloid in both human and animal tissues.     

Congo Red as a Simple Stain for Beta Cells of the Hypophysis

Bennhold's Congo red method for amyloid has been found to provide a simple, one step, differential staining technique for cells of the anterior lobe of the hypophysics, and to be applicable to routine use with formalin-fixed tissue. The beta cells stain orange-red and the alpha cells yellow, while the chromophobe cytoplasm remains unstained. This method can be used on material from humans and laboratory animals. Ordinary degrees of post-mortem change do not affect the staining reaction.     

Congo Red as a Simple Stain for Beta Cells of the Hypophysis

Bennhold's Congo red method for amyloid has been found to provide a simple, one step, differential staining technique for cells of the anterior lobe of the hypophysics, and to be applicable to routine use with formalin-fixed tissue. The beta cells stain orange-red and the alpha cells yellow, while the chromophobe cytoplasm remains unstained. This method can be used on material from humans and laboratory animals. Ordinary degrees of post-mortem change do not affect the staining reaction.     

Evidence that supramolecular Congo red is the sole ligation form of this dye for L chain lambda derived amyloid proteins.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.     

Alzheimer's disease: immunohistochemical characterization of cerebral amyloid deposits

In Alzheimer cortex tissue sections, thioflavine stained three patterns of amyloid lesions: neurofibrillary tangles (NFT), senile plaques (SP) and vessel walls (amyloid angiopathy AA). An anti serum against Tau proteins detected NFT but neither SP nor AA. In contrast, an anti serum against beta protein amyloid (BP A4) revealed SP and AA but not NFT. A periodic acid pretreatment dramatically enhanced the anti-BP A4 immunolabelling corresponding to microplaques as well as a large amount of diffuse extracellular amyloid substance, but never stained NFT. Pretreatment of tissue sections with a mixture of endo and exoglycosidases gave identical results and corroborates the extraneuronal processing of BP A4 that appears in a glycosylated form in the extracellular compartment.     

Copper iodide staining of protein blots on nitrocellulose membranes

Copper iodide staining which can detect protein levels as low as 100-150 pg/mm2 on nitrocellulose membranes is described. The staining is quantitative as measured by densitometry. Staining is complete within 5 min and may be removed by washing the membrane for 15 min without loss of immunoreactivity. The stain utilizes a reddish-brown precipitate of copper iodide in highly alkaline conditions. Because of its high sensitivity, convenience, and low cost, this stain may be more practical than amido black or gold- and silver-based stains for most laboratory purposes.     

A molecular history of the amyloidoses

The molecular investigation of the amyloidoses began in the mid-19th century with the observation of areas in human tissues obtained at autopsy that were homogeneous and eosinophilic with conventional stains but became blue when exposed to mixtures of iodine and sulfuric acid. The foci corresponded to regions formerly identified as "waxy" or lardaceous. Subsequent identification of the characteristic staining of the same tissues with metachromatic dyes such as crystal violet or with the cotton dye Congo red (particularly under polarized light) and thioflavins allowed the pathological classification of those tissues as belonging to a set of disorders known as the amyloidoses. Not unexpectedly, progress has reflected evolving technology and parallel advances in all fields of biological science. Investigation using contemporary methods has expanded our notions of amyloid proteins from being simply agents or manifestations of systemic, largely extracellular diseases to include "protein-only infection," the concept that "normal" functional amyloids might exist in eukaryotes and prokaryotes and that aggregatability may be an intrinsic structural price to be paid for some functional protein domains. We now distinguish between the amyloidoses, that is, diseases caused by the deposition of amyloid fibrils and amyloid proteins (i.e., purified or recombinant proteins that form amyloid fibrils in vitro), which may or may not be associated with disease in vivo.     

Isolation of Low-Molecular-Weight Proteins from Amyloid Plaque Fibers in Alzheimer''s Disease

During aging of the human brain, and particularly in Alzheimer's disease, progressive neuronal loss is accompanied by the formation of highly stable intra- and extraneuronal protein fibers. Using fluorescence-activated particle sorting, a method has been developed for purifying essentially to homogeneity the extracellular amyloid fibers that form the cores of senile plaques. The purified plaque cores each contain 60-130 pg of protein. Their amino acid composition shows abundant glycine, trace proline, and approximately 50% hydrophobic residues; it resembles that of enriched fractions of the paired helical filaments (PHF) that accumulate intraneuronally in Alzheimer's disease. Senile plaque amyloid fibers share with PHF insolubility in numerous protein denaturants and resistance to proteinases. However, treatment of either fiber preparation with concentrated (88%) formic acid or saturated (6.8 M) guanidine thiocyanate followed by sodium dodecyl sulfate causes disappearance of the fibers and releases proteins migrating at 5-7,000 and 11-15,000 Mr which appear to be dimerically related. Following their separation by size-exclusion HPLC, the proteins solubilized from plaque amyloid and PHF-enriched fractions have highly similar compositions and, on dialysis, readily aggregate into higher Mr polymers. Antibodies raised to the major low-Mr protein selectively label both plaque cores and vascular amyloid deposits in Alzheimer brain but do not stain neurofibrillary tangles, senile plaque neurites, or any other neuronal structure. Thus, extraneuronal amyloid plaque filaments in Alzheimer's disease are composed of hydrophobic low-Mr protein(s) which are also present in vascular amyloid deposits. Current evidence suggests that such protein(s) found in PHF-enriched fractions may derive from copurifying amyloid filaments rather than from PHF.     

Selective Staining of Eosinophils and Their Immature Precursors in Tissue Sections and Autoradiographs with Congo Red

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Notes on Technic: A Carbonate-Colloidal Iron Staining Reagent

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Displacement

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic tests Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack of contrast between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which doer have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acidophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.