Acknowledgments

Special thanks to friends, colleagues and patients at the National Amyloidosis Centre for their support and encouragement.

Introduction

History

Amyloidosis is a disorder of protein folding, in which normally soluble proteins misfold and aggregate in a characteristic highly ordered fashion, and are deposited in the extracellular space as insoluble fibrils (or filaments). These interstitial fibrillar protein deposits (generically known as amyloid) may form anything from microscopic plaques to confluent masses. Amyloid can progressively replace the parenchyma of affected organs, which may become significantly enlarged, damaging the tissue structure and function. This eventually leads to organ failure, often resulting in death.

The term amyloid means ‘starch-like’ and was first used to describe the starchy cellulose material found in plants, giving a deep blue/violet color with iodine. This reaction was adapted by who found that corpora amylacea of the brain had the same tinctorial properties as starch. He went on to investigate tissues containing ‘real’ amyloid, which he found also gave the same coloration. To date, amyloid continues to be defined and identified by its characteristic histological staining reactions.

The first molecular investigations began in the mid-19th century with the observation at autopsy that the cut surface of amyloid-affected organs may show a ‘waxy’ texture, often described by early pathologists as ‘waxy degeneration’ or ‘lardaceous disease’ ( ). Throughout this period this appearance was a frequent post-mortem finding in patients suffering from chronic inflammatory diseases such as tuberculosis (TB), although amyloidosis itself was rarely diagnosed during the patient’s lifetime.

At that time the exact composition of amyloid was unknown, other than it had a proteinaceous/albuminous nature with a high nitrogen content. As techniques improved during the twentieth century, a consensus was made that amyloid was predominately composed of proteins with 1–5% mucopolysaccharide present.

With the development of the textile industry in the 1880s, dyes became readily available and used for many applications other than textiles, including histological techniques. used the paper dye Crystal violet and the cotton dye Congo red for the demonstration of amyloid. noted the dichroic effect of Congo red stained-amyloid when viewed under polarized light. Subsequently, in , the optical activity of Congo red-stained amyloid giving the unique ‘apple-green birefringence’ was described by Divry & Florkin. This phrase is still in use today to describe the birefringence and dichroism that amyloid displays when viewed under crossed polarized light, although suggested that the correct phrase should be of ‘anomalous colors’, describing all the colors amyloid stained with Congo red can produce. With the advent of electron microscopy in the 1950s it was observed that amyloid had a unique fibrillary ultrastructure independent of anatomical site; quite different to any other ultrastructural fibrils described previously. In , Eanes and Glenner used X-ray diffraction to reveal that the protein within amyloid fibrils was arranged in an anti-parallel β-pleated sheet ( Fig. 15.1 ).

Fig. 15.1, Schematic diagram of amyloid structure.

Since then it has been shown that all amyloid fibrils share a common β-core structure with polypeptide chains running perpendicular to the fibril long axis, regardless of the particular protein from which they are formed ( ).

Composition

Following the development of techniques enabling amyloid fibril isolation ( ) it was confirmed that the bulk of amyloid deposits were composed of protein. They also contain up to 15% of a non-fibrillary glycoprotein known as amyloid P component (AP), derived from, and identical to the normal circulating plasma protein serum amyloid P (SAP). SAP is a calcium-dependent, ligand-binding protein which also forms a normal component of basement membranes and elastic fibers, and may have a function related to its binding to glycosaminoglycans (GAGs), fibronectin and other cellular components ( ). SAP belongs to the pentraxin family, and becomes specifically and highly concentrated in amyloid deposits of all types. This binding specificity to amyloid fibrils is the basis for radiolabeled SAP scintigraphy in patients with amyloidosis, a diagnostic technique which also enables quantitative monitoring of amyloid deposits ( ). The generic SAP ligand on amyloid fibrils remains uncharacterized ( ).

The composition of the GAGs includes various sulfates, heparan, chondroitin and dermatan, which may be involved in amyloidogenesis. The presence of the carbohydrate moieties of GAGs provides a possible explanation for the staining reaction in some of the histological methods used for amyloid detection.

Ultrastructure

Electron microscopy (EM) played an important role in the identification of the composition of amyloid, showing its unique fibrillary arrangement. To this day, EM is one of the methods for identifying amyloid. Amyloid deposits appear as masses of extracellular, non-branched filaments, usually in a random orientation (though occasionally in parallel arrays of a few fibrils). Each fibril consists of two electron-dense filaments 2.5–3.5 nm in diameter, separated by a 2.5 nm space, giving a total diameter of 8–10 nm with variable length of up to several μm ( ; ) ( Fig. 15.2a, b ).

Fig. 15.2, (a) Electron micrograph of kidney from a case of amyloidosis with renal involvement showing amyloid (A), epithelial cytoplasm (Epi C), basement membrane (BM) and an endothelial cell (Endo C). (b) A higher magnification of Fig. 15.2a where amyloid fibrils can be clearly seen.

Classification and nomenclature

There have been many attempts to identify and classify amyloid proteins in order to collate the endless variety of clinical manifestations, histopathological appearances and associated pathology. Until 1980, traditionally the main classification used for amyloid was that of , who divided amyloid types into four categories:

  • 1.

    Primary amyloid occurring spontaneously in the absence of an apparent predisposing illness. It affects organs and tissues such as heart, muscle, skin and tongue.

  • 2.

    Secondary amyloid occurring in patients with chronic infective diseases, e.g. syphilis and TB. Later, inflammatory diseases such as rheumatoid arthritis were also included in this group.

  • 3.

    Tumor-associated amyloid.

  • 4.

    Myeloma-associated amyloid.

As more and more amyloid-forming proteins were identified, this classification became obsolete. In 1974, a committee was organized at the international symposium on amyloidosis in Finland to oversee the nomenclature, while in , Husby et al. proposed guidelines for a better classification based on the identity of the amyloid fibril protein. This classification was adopted by the World Health Organization-International Union of Immununological Societies (WHO-IUIS). Today, this forms the currently accepted classification of amyloid. The International Society of Amyloidosis (ISA) meets biennially and at each symposium reviews any new types and possible new nomenclature.

At present, there are 36 different proteins which have been accepted as major amyloid-fibril proteins ( Table 15.1 ). Amyloidosis nomenclature uses the letter A to designate amyloid, followed by an abbreviation of the name of the fibril protein. For example, immunoglobulin light chain amyloid protein is called AL (A + immunoglobulin light chain); this can be further added to by kappa (κ) or lambda (λ) subtype (e.g. ALλ type).

Table 15.1
Unrelated amyloid-forming proteins
Abbreviation Protein precursor Dominant tissues affected Amyloid type
AA Serum amyloid A protein Spleen, gut Reactive systemic AA amyloidosis
AL (κ & λ) Immunoglobulin light chain Any tissue except the brain parenchyma Systemic AL amyloidosis or localized AL amyloidosis
AH Immunoglobulin heavy chain Renal Systemic AL amyloidosis or localized AL amyloidosis
ATTR Variant transthyretin (prealbumin) Peripheral and/or autonomic nerves, cardiac, gastro-intestinal Familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC)
ATTRwt Wild-type transthyretin (prealbumin) Cardiac TTR not hereditary
AApoAI Variant apolipoprotein AI Kidney, liver Hereditary systemic amyloidosis
AApoAII Apolipoprotein AII Kidney, liver Hereditary
AApoAIV Apolipoprotein AIV Kidney, liver Sporadic aging amyloidosis
AApoCII Apolipoprotein CII Kidney Hereditary
AApoCIII Apolipoprotein CIII Kidney Hereditary
AGel Gelsolin Kidney Hereditary
ACys Cystatin C Brain Hereditary
Amyloid β-protein precursor Brain Alzheimer’s disease, Down’s syndrome, cerebral amyloid angiopathy
2 M β 2 -Microglobulin Kidney, prostate Dialysis-associated amyloid, corpora amylacea
APrP Prion protein Brain Creutzfeldt-Jacob disease (CJD), prion disease
ACal Calcitonin Thyroid Medullary carcinoma of thyroid-associated disease
AANF Atrial natriuretic factor Heart Cardiac amyloid
AIAPP Islet amyloid polypeptide (Amylin) Pancreas Type II diabetes amyloid, insulinoma
AFib Fibrinogen A α-chain Kidney Hereditary
ALys Lysozyme Kidney Hereditary
APro Prolactin Pituitary Aging amyloid
AIns Insulin Skin / soft tissue Amyloid derived from insulin at injection sites
ABri ABriPP Brain Familial dementia (British)
ADan A Dan PP
The product of the same gene ABri
Brain Familial dementia (Danish)
AMed Lactadherin Arteries Aging amyloid in arteries
ALac Lactoferrin Cornea Corneal amyloidosis
AKer Kerato-epitheliun Eyes Familial corneal amyloidosis
ALect2 Leukocyte common antigen 2 Kidney, liver LECT 2 amyloidosis
AGal7 Galectin 7 Skin Localized
ACor Corneodesmosin Cornified epithelia, hair follicles Localized
AOAPP Odontogenic ameloblast-associated protein Odontogenic tumors Localized
ASem1 Semnogelin 1 Seminal gland Localized
AEnf Enfuritide Skin, soft tissue Iatrogenic, injection sites
ASpc Lung surfactant protein Lung Localized
AαSyn α-Synuclein Central nervous system Acquired
ATau Tau Central nervous system Acquired

One of the 14 forms of amyloid proteins which can give rise to systemic disease.

Pathogenesis

The processes which cause proteins to become involved in amyloid formation, i.e. the conversion from normal functioning proteins into inert amyloid deposits, is the focus of much research. There is little in common between many different types of proteins involved ( ). Many protein misfolding and amyloid aggregation diseases are strongly associated with aging, and diseases such as Alzheimer’s and Parkinson’s disease are becoming among the most common debilitating medical conditions throughout the world today. Whilst the close association between the appearance of amyloid deposits and the onset of pathological events is well described, the specific mechanisms underlying these events is still not understood ( ). In certain amyloid types, the fibrils contain only a limited cleaved portion of the amyloid protein precursor, as is the case in Alzheimer’s disease.

Some amyloid fibril precursor proteins are rich in β-pleated sheet conformation in their native form, whereas others e.g. the prion protein, contain no β-pleated sheet and it is the de novo formation of β-pleated sheets which represent a fundamental pathological event in prion diseases. In some types of amyloidosis the whole precursor protein may be involved, or there may be proteolysis of the precursor protein with liberation of a smaller amyloidogenic fragment, as with AβPP. There could also be a mixture of two. In transthyretin (TTR) amyloidosis, the circulating protein is a tetramer, and a vital pathological event appears to be release of the monomer.

Amyloidosis is now considered to belong to the category of conformational diseases. The pathological protein aggregation reflects, at least in part, limited stability of the normal physiological conformation, with a propensity to adopt an alternative pathologic conformation. It has been proposed that such a grouping helps to provide an understanding of the etiology of these diseases. This opens the prospect for common approaches to therapeutic stratagems in a similar way that recognition of bacteria as the causative agents of many infections gave rise to the use of antibiotics being useful in such conditions, or of steroid therapy being of potential use for all inflammatory disorders ( ). In this context, it is interesting to note the development of ‘designer’ peptides which bind to Aβ and to prion proteins with the aim of preventing, and even reversing the conformational changes responsible for these respective disease processes ( ). This concept is becoming increasingly accepted, and it is becoming evident that amyloid is but one, albeit definable, subgroup within a larger group of misfolded or altered protein deposits which are associated with human disease ( Table 15.2 ).

Table 15.2
Protein conformation diseases
Conditions Affected proteins Associated diseases
Amyloidosis 36 known in humans ( Table 15.1 )
Serpinopathies α 1 -Antitrypsinneuroserpin α 1 -Antitrypsin storage disease
Hemoglobinopathies Hemoglobulin Sickle cell anemia
Drug and aging
induced inclusion body hemolysis
Lewy body diseases α-Synuclein Parkinson’s disease
Neuronal inclusion bodies Tau Alzheimer’s disease
Pick’s disease
Progressive supranuclear palsy
Superoxide dismutase Motor neuron disease
Ferritin Familial neurodegenerative disorder
Hirano bodies Actin Alzheimer’s disease
Polyglutamine repeats Huntingtin Huntington’s disease
Ataxin Spinocerebellar ataxias
Androgen receptor Spinomuscular atrophies
Prion diseases Prion protein CJD
Variant CJD
Gerstmann-Straussler-Scheinker disease
Kuru
Fatal familial insomnia
Japanese cerebral amyloid angiopathy (CAA)

Amyloidosis

In AL amyloidosis, previously known as ‘primary amyloidosis’, monoclonal immunoglobulin light chains, produced by acquired clonal plasma cell or other B-cell dyscrasias, form amyloid deposits widely throughout the tissues. These can be either of kappa (κ) or lambda (λ) isotypes. Systemic AL amyloidosis is the most common form of clinical amyloid disease in developed countries and causes the most fatalities. In systemic amyloidosis, deposits can be present in any or all of the viscera, connective tissues and blood vessel walls, although intracerebral amyloid deposits are never found ( ).

AL amyloidosis can also be localized, i.e. restricted to a particular organ or tissue, and usually has a benign prognosis. In the skin, the deposits cause benign lumps which can be excised or left untreated. Localized amyloid deposits are also common in the bladder and pulmonary tissue where they can cause obstruction, leading to complications.

There are five possible types of heavy chain amyloidosis (AH); G, A, D, E and M – these are rare.

AA amyloidosis, previously known as ‘secondary amyloidosis’, is a complication of a prolonged chronic infection and/or inflammatory condition. AA amyloidosis is consequent on a longstanding acute phase response, in which production of serum amyloid A protein (SAA) is greatly increased. SAA is an apolipoprotein produced in the liver. It is an acute phase protein which is synthesized at increased levels in patients with diseases such as rheumatoid arthritis, TB, Crohn’s disease, familial Mediterranean fever (FMF) and other hereditary periodic fevers.

Due to therapeutic developments in many inflammatory diseases over the last 20 years, the incidence of AA amyloidosis has greatly decreased. However, there are now cases associated with intravenous drug abuse and HIV infection. In approximately 6% of cases of AA amyloidosis, the underlying inflammatory disorder cannot be characterized ( ). Abnormally increased production of SAA over a long period is a prerequisite for development of AA amyloidosis.

There are various types of hereditary systemic amyloidosis which involve many different organ systems. They are difficult to treat and often fatal. This group of disorders are dominantly inherited and rare, but there are various clusters around the world. One of the most common types is due to point mutations in the TTR gene; there are approximately 10,000 affected individuals worldwide. With more than 100 amyloidogenic mutations in the TTR gene, the major features of hereditary TTR amyloidosis include severe, ultimately fatal peripheral and/or autonomic neuropathy (familial amyloid polyneuropathy, FAP), and/or cardiac amyloidosis. Hereditary amyloidosis is also associated with mutations in the genes encoding apolipoproteins AI and AII, fibrinogen α-chain, gelsolin, lysozyme, cystatin C and β-protein.

Although all forms of hereditary amyloidosis are inherited dominantly, the penetrance and expressivity are remarkably variable. Thus there may be marked differences in age of onset, amyloid deposition and clinical presentation, not only between families but also within families with the same mutation. In contrast to AA amyloidosis (in which the concentration of the amyloid fibril protein SAA is raised but of normal structure), AL and hereditary amyloidosis are associated with proteins which have abnormal structure conferring an inherent propensity to undergo aberrant folding rich in β-pleated sheets to form amyloid fibrils.

Leukocyte cell-derived chemotactin 2 (ALECT2) amyloidosis was discovered by whilst characterizing amyloid co-existing in a clear cell carcinoma nephrectomy specimen. When studying proteins with leukocyte chemotactic activity found LECT2 to stimulate chondrocytes and osteoblasts. However, the pathogenesis of LECT2 amyloid remains to be understood. Whilst there is no evidence that ALECT2 is an inherited condition, the disease has a strong ethnic bias with most patients being Hispanic, Mexican, Punjabi and Native American ( ). ALECT2 amyloid deposits predominantly affect the liver and kidneys.

Wild type transthyretin (ATTRwt) amyloidosis, previously referred to as senile systemic amyloidosis, is not hereditary, the precursor protein being normal plasma transthyretin. ATTRwt amyloid deposits can be found in the myocardium of up to approximately 25% of elderly subjects, with men being more affected than women. However, cardiac deposition sufficient to cause clinical disease is apparently rare, or perhaps under-diagnosed. Deposition of wild type transthyretin also occurs in other anatomical sites, including the prostate, bladder and blood vessel walls, but only occasionally with clinical consequences.

Other diseases in which amyloid occurs

As outlined by , amyloid is a histological feature of Alzheimer’s disease and type 2 diabetes mellitus but, unlike systemic amyloidosis, it is not known whether the amyloid causes these diseases. In Alzheimer’s there is an abundance of intracerebral amyloid deposits composed of β-protein, but there is poor correlation between the quantity of amyloid and the cognitive impairment. However, mutations which result in abundant deposition of β-protein as amyloid may result in early-onset Alzheimer’s disease.

In patients with type 2 diabetes, amyloid is frequently found in the pancreatic islets of Langerhans (IAPP), though this is not universal in all islets or in all patients with type 2 diabetes. As well as this, in diabetic patients amyloid can also be found at the site of insulin injection; as a result of the injected insulin (AIns), this causes a localized cutaneous lump. Insulin is known to be able to convert into a fibrillary form in vitro when subjected to certain physical or chemical stimuli such as heat or acidity ( ).

It is also frequently cited that transmissible spongiform encephalopathy (TSE) is an example of amyloidosis, although amyloid deposits are not necessarily present in the brains of patients with the disease, nor in cows with bovine spongiform encephalopathy (BSE).

There are other protein misfolding disorders characterized by abnormal aggregates of proteins which are incorrectly described as amyloid-related, e.g. Parkinson’s disease associated with Lewy bodies and the polyglutamine repeats which cause Huntington’s disease.

Another protein deposition disease, often confused with amyloid, is light chain deposition disease (LCDD), which may be mistaken histologically due its accumulation in the extracellular space. However, LCDD deposits lack the affinity for Congo red stain and do not produce the characteristic green birefringence of amyloid under cross-polarized light. Under EM, LCDD deposits are granular, which aids the distinction ( ).

Corpora amylacea (CA) or ‘false amyloid’ is not a disease as such but small benign masses which are composed of bundles of hyaline fibrils having the same Congo red staining properties as amyloid. CA is found as luminal concretions in prostate, lung and uterus.

Diagnosis

There is now an increased awareness of amyloidosis and more patients are being diagnosed and referred to central amyloid management units such as the National Amyloidosis Centre (NAC) in the UK.

However, some patients are still overlooked. The diagnosis requires the presence of amyloid in a tissue. The gold standard technique is Congo red histology, although EM may aid diagnosis. Biopsies are usually taken to investigate organ dysfunction e.g. of the kidneys in nephrotic patients, or of sural nerves in familial polyneuropathies. Amyloid is present in up to 90% of rectal and/or subcutaneous fat biopsies in systemic AA or AL amyloidosis; rectal biopsies or fine needle aspirates of subcutaneous tissue used to be the main method of screening ( ; ). Techniques have now improved greatly and cardiac biopsies, after a suggestive echocardiogram, are becoming more popular. It must be noted that a negative biopsy does not exclude the possibility of amyloidosis. In rectal biopsies, amyloid is usually found in the walls of submucosal vessels, so if the full thickness of the muscularis is not obtained the deposits will go undetected.

The use of SAP scintigraphy allows in vivo diagnosis as well as the monitoring of progression and regression of the amyloid deposits with treatment ( ). Unfortunately, SAP scintigraphy is unable to visualize amyloid within cardiac tissue because the heart is a moving organ. The bone scanning method DPD scintigraphy ( 99m Tc-3,3-diphosphono-1,2-propanodicarboxylic acid) was serendipitously discovered to have high affinity for cardiac ATTR amyloid, and is routinely performed as a method for imaging cardiac involvement in this type of amyloidosis ( ).

Differentiation between different amyloid types

With the recognition that different proteins form amyloid and are associated with different clinical syndromes it became necessary to identify particular fibril types histologically. As the treatment of amyloidosis is entirely type-specific, the correct identification of the fibril type is indispensable in clinical practice.

Methods of section pretreatment using trypsin or potassium permanganate before Congo red staining were devised ( ). After such pretreatment some amyloids lose their affinity for Congo red, most notably AA amyloid, whereas AL amyloid is resistant. These methods were always equivocal in practice and have been rendered obsolete by the use of immunohistochemistry and other techniques to identify the particular amyloid fibril protein specifically and reliably.

It is vitally important to discriminate between AA, AL and hereditary amyloidosis, as their treatments are entirely different. Therapy for patients with AA amyloidosis involves measures to reduce SAA production by treating the cause of the underlying inflammation. AL treatment is aimed at ablating the B-cell clone responsible for the amyloidogenic free light chain production using cytotoxic drugs. Hereditary amyloidosis treatment may ultimately result in organ transplantation.

The tools available today to differentiate amyloid type include direct assessment of the fibril type by immunohistochemistry, proteomics and, occasionally, fibril sequencing. Indirect investigations aimed at identifying the disorders associated with amyloidosis include searches for monoclonal immunoglobulins using conventional electrophoresis, immunofixation assays of serum and urine, the serum free light chain assay, serum assays for SAA, and sequencing of genes known to be associated with hereditary amyloidosis.

Demonstration

In hematoxylin and eosin (H&E) stained sections amyloid appears as an amorphous, eosinophilic, extracellular, faintly refractive substance which sometimes displays green birefringence under polarized light. However, it should be noted that collagen also has this appearance under polarized light in H&E-stained sections. Amyloid can also be weakly birefringent using a powerful light source when stained with periodic acid-Schiff. Whilst large deposits of amyloid can be identified in H&E-stained sections, small deposits, e.g. in vessels and rectal or bone marrow samples may be missed.

Congo red was developed as the first direct cotton dye in 1884, and has been ‘re-invented’ many times in the search for an optimal method for the detection of amyloid ( ). As with most histopathological methods, they are often performed on tissues which have been formalin fixed and processed into paraffin wax. Samples left standing in fixative for long periods of time may cause stains to be less sensitive and intense. Control sections must be used in all staining methods, and to demonstrate amyloid they should be cut when needed since they can lose reactivity if stored for longer than a year.

Congo red

The molecular formula for Congo red is C 32 H 22 N 6 Na 2 O 6 S 2 ( Fig. 15.3 ). It is a symmetrical sulphonated azo dye containing a hydrophobic center with two phenyl groups bound by a diphenyl bond to give a linear molecule which is largely hydrophobic ( ). Congo red is also a fluorescent dye ( ), although not specific for amyloid. Two factors are important to the Congo red-amyloid reaction; the linearity of the dye molecule and the β-pleated sheet configuration. If the spatial configuration of either is altered, even though the chemical groupings are left intact, the reaction fails. Furthermore, the Congo red mediated positive birefringence of amyloid implies that the dye molecules are arranged in a parallel fashion ( ). Recent work confirms the long-held belief that the Congo red molecule intercalates between two protein moieties at the interface between two adjacent antiparallel β-pleated sheets by disrupting the hydrogen bonds which are responsible for maintaining the β-sheet polymer, yet allowing maintenance of the integrity of the structure by the formation of new hydrogen bonds between protein and dye ( ).

Fig. 15.3, Chemical structure of Congo red dye.

Since its introduction as a histological stain by , Congo red staining of amyloid producing green birefringence when viewed under polarized light has become the diagnostic gold standard for this disorder. Although it was many years before the exact staining mechanism was understood, it is now well established that staining of amyloid by Congo red is due to hydrogen bonding between the Congo red dye and the β-pleated sheet in a highly oriented linear and parallel manner on the amyloid fibrils ( ). Any tissue component which binds Congo red in a linear way also exhibits green birefringence in polarized light; dense collagen fibers also bind Congo red dye in this fashion, and various other formalin-fixed tissues can be stained. By using an alkaline Congo red method this phenomenon is reduced ( ). However, Romhányi used 1% aqueous Congo red and claimed that if the tissue sections were mounted in gum arabic this problem is overcome. adapted Romhányi’s original method, using a long deparaffinization step of up to 5 days, together with a longer incubation in Congo red. This technique has shown that the amyloid has a stronger affinity to Congo red and therefore can be seen as more sensitive and selective. Many different tissue structures will also stain with 1% aqueous Congo red, and so it must be used under strict conditions using known amyloid-positive sections as controls.

The specificity of Congo red staining of amyloid can also be increased by using an alcoholic method combined with high ion strength and high pH. The Puchtler method combines all these aspects, giving a superior technique to demonstrate amyloid and green birefringence under polarized light.

A recent comparison of several Congo red staining methods made during a run of the UK NEQAS histology external quality control scheme found that Highman’s method gave the highest scores. At the NAC a significant percentage of biopsies which are referred each year have been reported incorrectly, with both false positives (5%) and false negatives (8%) ( ). This can have serious repercussions for patients’ treatment. Consequently, the NAC recommends the Puchtler method since the lack of a differentiation step means there is less intervention by the operator. It should be noted that false positives and false negatives can be caused by other factors even in this technique, e.g. if the correct section thickness is not used.

Alkaline Congo red technique ( )

The method obviates the need for a differentiation step by the inclusion of a high concentration of sodium chloride. This reduces background electrochemical staining whilst enhancing hydrogen bonding of Congo red to amyloid, resulting in a progressive and highly selective technique. The solutions should be discarded after 1 month. Ready to use commercial reagents and kits are available with a shelf life of up to 12 months.

Fixation

Not critical.

Stock solutions

1% aqueous sodium hydroxide

Stock solution a

Saturated sodium chloride in 80% ethanol.

Stock solution b

Saturated Congo red in 80% ethanol saturated with sodium chloride.

Leave to stand overnight before use.

Working solutions

  • A.

    To 100 ml of stock solution a add 1 ml of 1% aqueous sodium hydroxide and filter.

  • B.

    To 100 ml of stock solution b add 1 ml of 1% aqueous sodium hydroxide and filter.

  • Prepare just before use.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here