• The term amyloid, meaning starch-like, is a misnomer coined by Virchow when he observed that amyloid deposits would stain blue with the iodine reaction, suggesting the presence of starch or cellulose.
• Amyloid – is a protein that has an alternation in its secondary structure which imparts a particular insoluble form, called the beta-pleated sheet conformation.• By definition, any protein deposits staining with Congo red and exhibiting green birefringence when viewed with polarized light are amyloid
Components of amyloid:A. Fibril protein:• proteins make up 85-90% of the amyloid weight• responsible for tissue dysfunction due to pressure effects
B. P Component:• makes up about 10% of the amyloid’s weight• found on all amyloid fibrils, except those associated with Alzheimer’s senile plaques.• stabilizes fibril amyloid protein and decreases their clearance• plays a role in some of the other special stains that can demonstrate amyloid (TFT)
C. Non-amyloid components:• Collagen, fibrin and reticulin• Part of the amyloid OR are trapped by the amyloid OR are part of the body’s repair process from the cellular destruction caused by the amyloid.• This is one of the reasons why connective tissue stains (e.g. trichromes, PAS) stain amyloid the same color as collagen
Classification of AmyloidosisProcess Type DesignationAcquired systemic Primary AL Secondary AA Familial AFOrgan limited Senile ASLocalized Endocrine AE
Important Systemic amyloidosis Amyloid protein Precursor Syndrome or involved tissueAL Immunoglobulin light Primary, Myeloma chain associatedAA Serum AA (SAA) Reactive, chronic inflammatory conditionAß2m ß2 microglobulin HemodialysisAA SAA Familial mediterranean feverATTR Transthyretin Familial amyloidotic polyneuropathiesATTR Transthyretin Systemic senile Amyloidosis
Important localized amyloidosis Amyloid protein Precursor Syndrome or involved tisssueAß Aß protein precursor Alzheimer’s disease (AßPP)APrP Prior protein Spongiform encephalopathyAcal Calcitonin Medullary carcinoma of thyroidAIAPP Islet amyloid Type II diabetes, polypeptide insulinomasAANF Atrial natriuretic factor Isolated atrial amyloid
• The progressive accumulation of extracellular protein fibrils can lead to cellular atrophy, ischemia, necrosis,and, ultimately, organ failure due to the effects of these accumulated fibrils on blood supply and normal cellular function
Demonstration of Amyloid• H & E – homogenous pale pink color• Van Gieson – yellow to yellow/brown• Iodine (Gram’s or Lugol’s) – mahogany brown turning into blue on treating with 10% sulphuric acid
• Light microscopy – postive staining with congo red together with a resultant green polorization color• Electron microscopy – typical fibrillar ultra structure of amyloid• X-ray diffraction – cross beta pleat structure
I. Metachromatic methods• Staining with 1% aqueous methyl violet for 5 minutes, followed by differentiation with 1 % acetic acid• Sections should be mounted in aqueous media of high sugar or salt content to prevent diffusion of the stain
Lendrum’s technique1. Bring sections to water2. Stain with 1% aqueous methyl violet for 3 minutes3. Differentiate in 70% formalin (controlling microscopically)4. Wash in running water for 1 minute5. Flood with saturated aqueous sodium chloride for 5 minutes6. Rinse in water and mount in corn syrupResults: Amyloid- Pink to red Other elements- Violet
II. Congo red methods• Specificity of methods of this type are dependent on ▫ Beta pleated sheet configuration of amyloid ▫ Linearity of the dye molecule• Congo red is a linear dye molecule. It binds to amyloid by hydrogen bonds
• The characteristic green polarization color wasn’t present in very thin or thick section – thickness of 5-10 micron was optimal• Sirius red – similar properties to congo red• Tissue sections containing amyloid AA protein, affinity for Congo red is lost after pretreatment with potassium permanganate
Bennhold’s Technique1. Bring sections to water2. Stain with Ehrlich’s haematoxylin for 20 mins3. Differentiate with 1% acid alcohol4. Wash in running water for 1 min to remove acid5. Stain with 1% aqueous Congo red for 20-30 mins6. Pour off stain and flood slide with a saturated aqueous solution of lithium carbonate; leave for 15 secs7. Differentiate in 80% alcohol until excess Congo red is removed8. Wash in running water for 10 mins9. Dehydrate, clear and mount in Canada balsam or synthetic resinResults Amyloid- Pink to red Nuclei- Blue
Alkaline Congo red technique(Puchtler, Sweat and Levine)• Advantage of not requiring differentiation ( Congo red is in alkaline solution)• The use of alcoholic solutions, high salt content, and high pH, as in the Puchtler Congo red method, greatly increase staining specificity for amyloid
• A saturated salt solution in alcohol at alkaline pH is used in both the dye solution and as a pretreatment of the tissue sections just before staining.• High salt content and alkaline pH are believed to depress dye ionization and electrostatic binding to nonamyloid structures. Saturation of the salt and dye solutions is very important
• Fixation: ▫ Best results are obtained after alcohol or Carnoy fixed tissues ▫ Formalin or Zenker –fixed tissues were found to stain better than with other techniques
• Reagents ▫ Alkaline salt solution. To 50 ml of 80% alcohol saturated with sodium chloride add 0.5 ml of 1% aqueous sodium hydroxide. Filter and use within 15 min. ▫ Stock stain solution. Use 80% alcohol saturated with Congo red and sodium chloride ▫ Staining solution. Add 0.5 ml of 1% aqueous sodium hydroxide to 50 ml of stock stain, filter and use within 15 mins
Method :1. Bring sections to water.2. Stain in haematoxylin for 5 minutes.3. Rinse well in distilled water.4. Pretreat in alkaline alcohol-salt solution for 20 min.5. Stain in alkaline Congo red solution for 20 min.6. Dehydrate rapidly in three changes of absolute alcohol.7. Clear and mount in synthetic resin.Results: Amyloid- deep pink to red Nuclei -blue Elastic -pale pink
This bone marrow section stained with the Puchtler Congo redprocedure reveals intense amyloid deposits in the bone marrowsample
This figure viewed with polarizing microscopy;amyloid deposits exhibit characteristic apple-green birefringence.
Problems Encountered With theCongo Red Stain ..
a) PROBLEM: Weakly Stained Tissues• APPEARANCE: Faint uptake of Congo red dye throughout the section• CAUSES: ▫ Tissue was fixed for a prolonged period in a formaldehyde-containing fixative. ▫ Cut sections were stored for a prolonged period. ▫ Solutions of Congo red are not stable in the presence of salt and alkali.
• SOLUTIONS: • Avoid storing tissues in formaldehyde-based fixatives for prolonged periods of time. • Cut the sections just before staining, or cut only as many control sections as can be used in a short period of time. • Seal the cut paraffin blocks to help preserve control tissue reactivity. • Prepare fresh working solutions just before use
Congo red staining in this section appearsindistinct, suggesting that the stain wasexcessively differentiated
Examination of the section with polarizedlight reveals numerous amyloid deposits
b) PROBLEM: Nonspecific Staining• APPEARANCE: Structures other than amyloid bind the Congo red; high background staining may make it difficult to distinguish true amyloid deposits if present in the tissue• CAUSES: Collagen, elastic fibers, and keratin may stain nonspecificallywith aqueous Congo red solution. The pH is not sufficiently alkaline.
SOLUTIONS:• Use polarizing microscopy to help distinguish connective tissue components (grey or silver )from amyloid deposits (green).• Rotating the slide on the stage during polarizing microscopy can help distinguish true amyloid deposits from connective tissue. When the slide is rotated, connective tissue fibers will lose the dichroism,while amyloid will not• Use the Puchtler Congo red method, because the high salt content of the prestain rinse and staining solutions tends to diminish nonspecific staining.• Avoid the use of Canada balsam mounting medium because it will fluoresce.
Tissue components in this Congo red stainedkidney section appear to be nonspecificallystained
Polarization of the Congo red-stainedsection reveals that most of the redstaining is not amyloid. Some apple-greenbirefringence is noted in the glomerulus atthe bottom of the image
C. PROBLEM: Incorrect Color of BirefringenceAPPEARANCE: Structures may exhibit yellow, red, or white dichroismCAUSE:• Section thickness may be incorrect; this artifact is especially likely in thin sectionsSOLUTION:• Ensure that sections are cut at 5 to 10 μm.COMMENT:Old (large) amyloid deposits will often display diminished birefringence.Smaller deposits in blood vessel walls, for example, may be more likely to demonstrate the characteristic apple green color.
Congo red staining in this section of kidney appears specific and well differentiated
When viewed with polarized light, amyloiddeposits appear red to yellow, not theexpected apple-green color; incorrectsection thickness is the most likely cause
Collagen, appearing white to silver in thispolarized, Congo red stained section,surrounds a characteristic apple-greenamyloid deposit.
D. PROBLEM: Precipitate on TissueAPPEARANCE: A red precipitate is randomly present throughout the tissueCAUSE:Salt solutions were prepared with isopropyl alcohol.SOLUTION:Ensure that the salt solutions are prepared with ethyl alcohol because the salt does not dissolve well in isopropyl alcohol and will deposit crystals on the section.
Although the amyloid is well demonstrated inthis section, close inspection reveals a redgranular precipitate caused by using isopropylalcohol instead of ethyl alcohol as the solvent forthe salt solutions
III. Toluidine blue methodStandard toluidine blue1. Bring sections to water.2. Stain in 1% toluidine blue in 50% isopropanol for 30 min at 37o C.3. Blot and place in absolute isopropanol for 1 min.4. Clear in xylene and mount.Results:Amyloid is distinguished by its dark polarization color
IV. Fluorescence techniques• Thioflavine T technique• Extremely sensitive technique although not specific because ▫ stained sections are not permanent and ▫ tissue components other than amyloid, including fibrinoid, keratin, intestinal muciphages, Paneth cells, zymogen granules, and juxtaglomerular apparatus, all stain with thioflavine T
• Attempts made to increase specificity ▫ Using acid solutions ▫ Including Magnesium chloride• Thioflavine S may be substituted for Thioflavine T
Method:1. Bring sections to water.2. Stain in alum-haematoxylin for 2 min to quench nuclear fluorescence. The haematoxylin does not need to be differentiated, or blued.3. Wash in water for a few minutes.4. Stain in 1% aqueous thioflavine T for 3 min.5. Rinse in water.6. Differentiate in 1% acetic acid for 20 min.7. Wash in water.8. Mount in Apathy’s medium.Results:Amyloid and mast cells fluoresce bright yellow when examinedusing a BG12 exciter filter and an OG4 or OG5 barrier filter.The finest deposits can be seen using a UG1 or UG2 exciter filterwith a colourless ultraviolet barrier filter
Amyloid stained with TFT in Kidney.Hit with 490 (blue) excitor wavelength. Amyloidfluorescing yellow
• Congo Red Fluorescence Congo red stained amyloid, even if it does not birefringe with polarization, will often fluoresce an orange color when viewed with the auramine-rhodamine fluorescence microscope wavelengths (540 nm excitor wavelength) and lenses. At the same time, connective tissue and/or other tissue components that like to autofluoresce with green light will usually exhibit a white to yellow color
Congo Red stained amyloid in kidney, .Hit with 490 (blue) excitor wavelength.Amyloid fluorescing orange
V. Alcian blue method• Introduced by Lendrum, Slidders and Fraser• Fixation in formal-saline is usually adequate
• The sodium sulphate-Alcian blue (SAB) method• Reagents 1. Acetic alcohol a) 95% ethanol – 45ml b) Distilled water – 45 ml c) Glacial acetic acid - 10 ml Prepare fresh for use 2. SAB solution a) 1% alcian blue in 95 % ethanol – 45ml b) 1% aqueous sodium sulphate decahydrate – 45ml c) Glacial acetic acid – 10ml Prepare from stock solutions and stand for 30 mins before use
Method1. Bring sections to water.2. Immerse in acetic alcohol for 1-2 min.3. Stain in SAB working solution for 2 hours.4. Transfer to acetic alcohol for 1-2 min.5. Wash in water.6. Alkalinize in 80% ethanol saturated with borax for 30 min.7. Wash in water8. Stain nuclei with Celestine blue-haemalum sequence and counterstain with Van Gieson.9. Dehydrate, clear and mount.Results: Amyloid, mast cells and some colloids- Green
VI. IMMUNOHISTOCHEMISTRY• There are limitations as to the use of immunohistochemistry for the demonstration of amyloid• Each has its own sequence of amino acids (proteins). Also, there are variations of amino acid sequences from person to person, even if they have the same type of amyloid.• It is usually necessary to use more than one IHC for amyloid concurrent with the Congo red stain, as well as other amyloid histology stains, if needed.